Starchless variety of pisum sativum having elevated levels of sucrose

ABSTRACT

The present invention relates to a  Pisum sativum  variety that contains a recessive gene called the bsg gene and produces peas that exhibit an elevated level of sucrose and a decreased level of alcohol insoluble solids when compared to peas produced from a  Pisum sativum  variety that does not contain the bsg gene within its genome.

RELATED APPLICATION INFORMATION

This application is a 371 of PCT/US98/25912 filed on Dec. 7, 1998, which claims priority from U.S. Ser. No. 09/015,711 filed on Jan. 29, 1998, now U.S. Pat. No. 6,127,605, which is a continuation-in-part of U.S. Ser. No. 08/986,616 filed on Dec. 8, 1997, now abandoned.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a Pisum sativum variety that contains a recessive gene that produces highly wrinkled seed having a low starch content. Additionally, the present invention relates to nucleotide sequences for said recessive gene and methods for using said nucleotide sequences in site-specific recombination.

BACKGROUND OF THE INVENTION

The garden pea (Pisum sativum L.) is a commercially important food crop and the immature seed of the garden pea (“peas”) are widely consumed. There are a large number of genes that affect starch or carbohydrate synthesis in peas. The first reported and best described gene is the r gene (see White, O. E., Proceedings of the American Philosophical Society, 56: 487-588). The r mutant is believed to have occurred spontaneously at the beginning of the seventeenth century (see Lamprecht, H., Agri. Hortique Genetica 14: 1-4 (1956)) and its mature, dry seed has a wrinkled appearance (hence “r”, derived from the Latin, rugosus meaning “wrinkled or shriveled”). Wrinkling of mature seed was one of the characteristics used by Mendel in experiments which led him to formulate his laws of inheritance. (see Mendel, G., Verhandlungen des Naturforschenden Vereinds in Brünn 4: 3-47 (1865)).

Recessive alleles at the r locus not only have a profound effect on the shape of the seed, but also have numerous effects at all levels of seed development. Wang, T. L., et al., Seed Science Research 1:3-14 (1991). Specifically, seeds of the r mutant contain a lower proportion of starch than the wildtype (about 30% dry weight as opposed to about 50%) with the starch composition being altered to contain a higher proportion of amylose and a small proportion of amylopectin (with about 70% of dry weight of the starch of mutant seeds being amylose as opposed to 38% of the wildtype starch). For a long period of time, the molecular basis of the mutation was not clear. The difference in the level and composition of starch in the seed led several investigators to examine the various enzymes of starch biosynthesis. Id. Early studies indicated that the mutation was likely to be in the starch branching enzyme and this was confirmed when it was shown, using a pair of lines near-isogenic except for genes at the r locus that rr embryos lacked one form of the starch-branching enzyme, which is frequently referred to as “SBE1”. Subsequently, it was shown that the mutant was caused by a transposon-like insertion in the gene encoding SBE1. Id.

A second recessive rugosus locus termed “rb” is also known. Mutants homozygous recessive at this locus exhibit a wrinkled seed phenotype similar to that of rr plants. However, the amount of starch and its composition in the rb mutant are different than that of the r mutant. Specifically, seed of the r mutant contains about 30% dry weight of starch, of which about 70% is amylose. Seed of the rb mutant contains about 36% dry weight of starch, of which about 23% is amylose. The rb mutation has been found to result in reduced activity in the enzyme ADP glucose pyrophosphorylase. Purification of the enzyme and western blot experiments have revealed the absence of one of the four polypeptide subunits present in the wildtype enzyme.

WO 98/01574 describes Pisum sativum varieties which contain a mutation referred to as “rug3”. Plants containing the rug-3 mutation produce wrinkled seeds having low levels of starch, high levels of sucrose, as well as a high protein and lipid content. The rug-3 mutation has been found to be associated with a reduction in the activity of the enzyme plastid phosphoglucomutase (hereinafter “PGM”). According to WO 98/01574, pea seeds containing the rug-3 mutation have a sucrose content of greater than 6% by weight of the total weight of the seed as harvested at a tenderometer reading exceeding 120 tenderometer units.

The rug-3 mutation described in WO 98/01574 was induced via a mutagenesis program. Twenty thousand phenotypically round genetically wildtype (RR) seeds (referred to as “M1” seed) were treated with ethyl methanesulphonate or N-methyl-N-nitrosourea, which are known mutagens. M1 seed gave rise to M1 plants bearing M2 seed. M2 seed gave rise to M2 plants bearing M3 seed. M3 seeds were analyzed for storage product content. Seeds which exhibited a wrinkled appearance were selected from the M3 generation. These seeds were found to contain a wide range of starch, from 0-60% as a proportion of the dry weight of the mature seed. Within the starch of these seeds, the amylose content ranged from 0-80%. Also, the lipid content of the peas ranged from about 1 to about 8% of the dry weight and protein ranged from about 24 to about 48%. WO 98/01574 does not contain any information characterizing the rug-3 mutant except to describe how this mutation was induced via a mutagen program. Additionally, WO 98/01574 does not contain any information that distinguishes the DNA sequence of the rug-3 mutant from that of the wildtype and contains no information on effects on alcohol insoluble solids content.

WO 98/01574 also describes the isolation and characterization of the nucleotide sequence for wildtype pea plastid phosphoglucomutase. According to WO 98/01574, this nucleotide sequence can be introduced into a plant via recombinant DNA technology to produce transgenic plants in which the plastid PGM gene expression is down regulated or inactivated in developing seeds.

Sweetness in peas, associated with increased sugar content, is generally prized by consumers, who perceive that sweeter peas have a better flavor. Thereupon, because peas are such an important food crop, there is a need in the art for peas having an increased sweetness.

SUMMARY OF THE INVENTION

The present invention relates to a new variety of Pisum sativum, which is resistant to Fusarium Wilt Fungus and Powdery Mildew Fungus and which contains within its genome, a homozygous recessive gene, referred to as the bsg gene. It has been determined that the bsg gene contains a mutation in an intron in a 3′ splice site of the gene. More specifically, the bsg gene has the genomic nucleotide sequence shown in Sequence ID NO:1 and contains a mutation at nucleotide 1548 at the 3′ splice site dinucleotide AG, where nucleotide A is replaced with nucleotide T.

A Pisum sativum variety that contains the bsg gene within its genome produces peas (known in the art as immature seeds) which exhibit a lower level of starch, an elevated level of sucrose and a decreased level of alcohol insoluble solids when compared with peas produced from a Pisum sativum variety that does not contain the bsg gene homozygous within its genome.

Additionally, the present invention contemplates a Pisum sativum variety which contains a homozygous bsg gene within its genome. The bsg gene has the nucleotide sequence shown in SEQ ID NO:1 and contains a mutation in an intron at nucleotide 1548 at the 3′ splice site dinucleotide AG, where nucleotide A is replaced with nucleotide T.

The peas of the present invention contain from about 6.0 to about 7.5 percent fresh weight of sucrose when measured at a tenderometer value of from about 90 to about 110 and from about 6.5 to about 8.0 percent by weight of alcohol insoluble solids when measured at a tenderometer value of about 105. Moreover, the peas of the present invention contain from about 5 to about 30 percent fresh weight more sucrose than peas produced from a Pisum sativum variety that does not contain the bsg gene homozygous within its genome. Additionally, the peas of the present invention exhibit twenty (20) percent less alcohol insoluble solids when compared with peas from a Pisum sativum that does not contain the bsg gene homozygous within its genome.

Additionally, the present invention relates to a process for producing peas of a Pisum sativum variety that contain higher levels of sucrose and lower levels of alcohol insoluble solids than peas from a Pisum sativum variety that does not contain the bsg gene homozygous within its genome. The said process involves crossing a Pisum sativum variety or line that contains the bsg gene homozygous within its genome with a second Pisum sativum variety or line that contains the bsg gene homozygous within its genome, collecting the resulting mature seeds, planting the mature seeds, growing the mature seeds into Pisum sativum plants, selecting Pisum sativum plants with desirable phenotypic traits; allowing the plants to self-pollinate until a uniform line is produced, allowing the Pisum sativum line to self-pollinate, selecting plants with desirable phenotypes and collecting the resulting peas (which are also referred to as “mature seeds”).

In another embodiment, the process involves crossing a Pisum sativum variety or line that contains the bsg gene homozygous within its genome with a second Pisum sativum variety or line which does not contain the bsg gene within its genome, collecting dry, mature seeds, planting the collected dry, mature seeds, growing the mature seeds into Pisum sativum plants, allowing the plants to self-pollinate, collecting the resulting dry, mature seeds, selecting highly wrinkled mature seeds that do not contain organized starch grains and which do not stain purple when treated with a solution of iodine and potassium iodide, planting said highly wrinkled mature seeds, growing the mature seeds into Pisum sativum plants, selecting plants with desirable phenotypic traits, allowing the plants to self-pollinate until a uniform Pisum sativum line is produced, allowing the Pisum sativum line selected to self-pollinate, and collecting the resulting peas. The Pisum sativum variety or line that does not contain the bsg gene within its genome can contain any combination of the genes such as the r, rb, R or Rb homozygous within its genome. The peas produced by the process of the present invention contain from about 6.0 to about 7.5 percent fresh weight of sucrose when measured at a tenderometer value of from about 90 to about 110 and from about 6.5 to about 8.0 percent by weight of alcohol insoluble solids when measured at a tenderometer value of about 105.

The present invention also contemplates a process of producing highly wrinkled mature seed of a Pisum sativum variety that contains the bsg gene within its genome. In one embodiment the process involves crossing a Pisum sativum variety or line that contains the bsg gene within its genome with a second Pisum sativum variety or line that contains the bsg gene within its genome and collecting the resulting mature seeds.

In another embodiment, the process involves crossing a Pisum sativum variety or line that contains the bsg gene within its genome with a Pisum sativum variety or line that does not contain the bsg gene within its genome, collecting mature seeds, planting the collected mature seeds, growing the mature seeds into Pisum sativum plants, allowing the plants to self-pollinate, collecting mature seeds, selecting highly wrinkled seeds that do not contain organized starch grains, planting said mature seeds and growing the seeds into Pisum sativum plants, selecting plants with desirable phenotypic traits, allowing the plants to self-pollinate until a uniform Pisum sativum line is produced, allowing the Pisum sativum line to self-pollinate and collecting the mature seeds.

The present invention also contemplates Pisum sativum varieties grown from the mature seed described above and peas harvested from said varieties.

In another embodiment, the present invention contemplates a DNA molecule containing a nucleotide sequence which encodes plastid phosphoglucomutase, where the nucleotide sequence contains at least one mutation in an intron. The mutation in the intron prevents the excision of the intron from a primary transcript during post-transcriptional modification of the transcript and produces at least one aberrant mRNA for translation. Preferably, the mutation is a mutation in a 3′ splice site in the intron. Most preferably, the mutation is in a dinucleotide AG, where the nucleotide A is replaced by nucleotide T. This DNA molecule can be isolated and purified and inserted into an expression vector containing the DNA molecule, a promoter and a polyadenylation signal. In this vector, the promoter is operably linked to the DNA molecule and the DNA molecule is operably linked to the polyadenylation signal. The promoter may be the cauliflower mosaic virus 35S promoter and the polyadenylation signal may be the polyadenylation signal of the cauliflower mosaic 35S gene. Bacterial cells, such as Agrobacterium tumefaciens and Agrobacterium rhizogenes cells, and plant cells may be transformed with this vector.

In yet another embodiment, the present invention relates to a plant which contains a gene encoding plastid phosphoglucomutase within its genome, where the gene contains at least one mutation in an intron. The mutation in the intron prevents the excision of the intron from a primary transcript during post-transcriptional modification of the transcript and produces at least one aberrant mRNA for translation. The plant containing such a mutated gene may be a monocot or a dicot. Preferably, the mutation is a mutation in a 3′ splice site in the intron. Most preferably, the mutation is in a dinucleotide AG, where the nucleotide A is replaced by nucleotide T.

In yet another embodiment, the present invention contemplates an isolated and purified DNA molecule having the nucleotide sequence shown in SEQ ID NO:1 as well as an expression vector containing this DNA molecule, a promoter and a polyadenylation signal. In this vector, the promoter is operably linked to the DNA molecule and the DNA molecule is operably linked to the polyadenylation signal. The promoter may be the cauliflower mosaic virus 35S promoter and the polyadenylation signal may be the polyadenylation signal of the cauliflower mosaic 35S gene. Bacterial cells, such as Agrobacterium tumefaciens and Agrobacterium rhizogenes cells, and plant cells may be transformed with this vector.

Finally, in yet another embodiment, the present invention contemplates a method of altering the level of plastid phosphoglucomutase expressed in a plant. The method involves transforming plant cells with a vector. The vector contains a promoter, a polyadenylation signal and DNA molecule having either (1) the nucleotide sequence of SEQ ID NO:1; or (2) a nucleotide sequence encoding plastid phosphoglucomutase where the nucleotide sequence contains at least one nucleotide which prevents the excision of at least one intron from a primary transcript during post-transcriptional modification of the transcript and which produces at least one aberrant mRNA for translation. The promoter is operably linked to the DNA molecule and the DNA molecule is operably linked to the polyadenylation signal.

The second step of the method involves regenerating plant cells to provide a differentiated plant. The final step of the method involves identifying a transformed plant having an altered level of plastid phosphoglucomutase expressed in that plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows starch granules from three different types of dry, mature Pisum sativum seeds. FIG. 1a shows the starch granules from a smooth seed which contain the R gene homozygous within its genome. FIG. 1b shows the starch granules from a wrinkled seed which contains a r gene homozygous within its genome. FIG. 1c shows the near absence of starch granules and the presence of granular material of unknown composition in mature seed containing a homozygous bsg gene.

FIG. 2 shows starch biosynthesis in a chloroplast.

FIG. 3 shows starch biosynthesis in an amyloplast.

FIG. 4 shows the electrophoretic separation of phosphoglucomutase activities from a pea variety containing the r gene homozygous within its genome and from a Pisum sativum variety containing the bsg gene homozygous within its genome. Lane 1 shows leaf extract from a variety containing the bsg gene. Lane 2 shows chloroplasts isolated from a variety containing the r gene. Lane 3 shows leaf extract from a variety containing the r gene. Lane 4 shows chloroplasts isolated from a variety containing the r gene. Lane 5 shows leaf extract from a variety containing the bsg gene.

FIG. 5 shows an electrophoretic separation of phosphoglucomutase activities from peas containing the r gene and from peas containing the bsg gene. Lane 1 shows a cotyledon extract from a variety containing the r gene. Lane 2 shows a cotyledon extract from a variety containing the bsg gene.

FIG. 6 shows part of the genomic sequence of plastid PGM from a Frimousse introgressed-bsg variety (hereinafter referred to as the “mutant”) (SEQ ID NO:1). Exons are shown in bold.

FIG. 7 shows part of the genomic sequence of the smooth-seed Pisum sativum variety called “Frimousse”(hereinafter referred to as the “wildtype”) (SEQ ID NO:2). Exons are shown in bold.

FIG. 8 shows a Northern blot analysis of RNA from wildtype and mutant plants hybridized with a plastid PGM probe. Lane 1 shows the total RNA from a wildtype cotyledon. Lane 2 shows total RNA from a mutant cotyledon. Lane 3 shows total RNA from a wildtype cotyledon.

FIG. 9 shows the nucleotide sequence of wildtype plastid phosphoglucomutase (reported in WO 98/01574) the primers PGM-Fp (CACTGTTACA GACTCGATCA ATGG-SEQ ID NO:3), PGM-F91 (CAGACTCGAC AACTTCATCA TCTC-SEQ ID NO:4), PGM-Fb (GATTGCTGAT ATTCCCGATG TTGA-SEQ ID NO:5), PGM-F827 (GACCCAGTTT CTGATTACCT GGAG-SEQ ID NO:6), PGM-Rg (CATGCATGGCATCAAATGTA AACC-SEQ ID NO:7), PGM-R1262 (GCATTGATCG TGCAAGACCCTTGA-SEQ ID NO:8), PGM-R1816 (CACTCTAACA GTTGCACCAG CAGA-SEQ ID NO:9) AND PGM-RF (TCCCTGTGAA GTCTTTGAGC TTTG-SEQ ID NO:10) which were used in PCR to amplify PGM cDNA from the wildtype and mutant poly(A)+seed.

FIG. 10 shows the PCR amplification of PGM plastid cDNA from the wildtype (represented by a “F”) and f (represented by a “Fm”) poly(A)+seed RNA.

FIG. 11 shows the nucleotide and corresponding amino acid sequence of plastid PGM cDNA from the wildtype (SEQ ID NOS:13 and 14).

FIG. 12 shows the nucleotide and corresponding amino acid sequence of plastid PGM cDNA1 from the mutant (SEQ ID NOS:15 and 16).

FIG. 13 shows the nucleotide and corresponding amino acid sequence of plastid PGM cDNA2 from the mutant (SEQ ID NOS:17 and 18).

FIG. 14 shows the nucleotide and corresponding amino acid sequence of plastid PGM cDNA3 from the mutant (SEQ ID NOS:19 and 20).

DETAILED DESCRIPTION OF THE INVENTION Sequence Listing

The present application also contains a sequence listing that contains 21 sequences. The sequence listing contains nucleotide sequences and amino acid sequences. For the nucleotide sequences, the base pairs are represented by the following base codes:

Symbol Meaning A A; adenine C C; cytosine G G; guanine T T; thymine U U; uracil M A or C R A or G W A or T/U S C or G Y C or T/U K G or T/U V A or C or G; not T/U H A or C or T/U; not G D A or G or T/U; not C B C or G or T/U; not A N (A or C or G or T/U)

The amino acids shown in the application are in the L-form and are represented by the following amino acid-three letter abbreviations:

Abbreviation Amino acid name Ala L-Alanine Arg L-Arginine Asn L-Asparagine Asp L-Aspartic Acid Asx L-Aspartic Acid or Asparagine Cys L-Cysteine Glu L-Glutamic Acid Gln L-Glutamine Glx L-Glutamine or Glutamic Acid Gly L-Glycine His L-Histidine Ile L-Isoleucine Leu L-Leucine Lys L-Lysine Met L-Methionine Phe L-Phenylalanine Pro L-Proline Ser L-Serine Thr L-Threonine Trp L-Tryptophan Tyr L-Tyrosine Val L-Valine Xaa L-Unknown or other

Description of the Pea Varieties Containing the bsg Gene

The present invention relates to a novel garden variety of pea, Pisum sativum, that contains within its genome, a homozygous recessive gene, referred to as “bsg” (bsg referring to “blown starch grain”). As used herein, the term “genome” refers to the entire hereditary material (DNA) in a cell which is composed of one or more chromosomes.

It is well known in the art that peas are the immature seeds of a Pisum sativum variety. Peas or immature seeds typically do not exhibit a wrinkled appearance, but rather a smooth, round appearance. These peas contain the R gene as the dominant allele. Peas or immature seeds are consumed by individuals as food. Mature seeds of a Pisum sativum variety are dry seeds. Mature seed exhibits a wrinkled appearance if it contains the r or rb genes or bsg gene within its genome. Mature seeds are used for planting and produce Pisum sativum varieties.

The Pisum sativum variety of the present invention contains a homozygous bsg gene within its genome and produces mature seeds which are highly wrinkled, have a very low starch content and high levels of sucrose. The mature seeds of the present invention also contain a homozygous bsg gene within their genome and contain few, if any, organized starch granules when examined under a microscope. Instead of containing starch granules, mature seeds of the present invention contain granular material of unknown composition.

FIG. 1 demonstrates the appearance under a microscope of starch granules in various types of mature Pisum sativum seeds. FIG. 1a shows the appearance of starch granules in smooth mature seeds that contain the R gene homozygous or heterozygous with their genome. These mature seeds have entire starch granules and do not contain the r or bsg genes homozygous within their genome. When the mature seeds shown in FIG. 1a are treated with a solution of iodine and potassium iodide, the mature seeds stain purple indicating the presence of starch. FIG. 1b shows the appearance of the starch granules in a wrinkled mature seed that contains the r gene homozygous within its genome. The starch granules in the wrinkled mature seed containing the r gene homozygous exhibit a star shaped fracture pattern similar to that of a pie that has been scored into 5 or 6 pieces. FIG. 1c shows mature seed of the present invention that contains the bsg gene homozygous within its genome. As shown in FIG. 1c, mature seeds of the present invention contain few, if any, organized starch granules but instead contain granular material. When the mature seeds shown in FIG. 1c are treated with a solution of iodine and potassium iodide, the mature seeds fail to stain purple due to the near absence of starch.

The bsg mutation of the present invention was not induced by a mutagen program like the rug-3 mutation described in WO 98/01574. Instead, the bsg-mutation of the present invention is a natural mutation which was observed in a crop of smooth seed of a Pisum sativum variety containing the R gene as the dominant allele.

Due to the absence of starch, the Pisum sativum variety of the present invention also produces the peas or immature seeds that contain elevated levels of sucrose. As used herein, the term “sucrose” refers to the disaccharide composed of glucose and fructose.

The peas of the present invention contain the bsg gene homozygous within their genome and further contain from about 6.0 to about 7.5, preferably from about 6.2 to about 7.1 percent fresh weight of sucrose when measured at a tenderometer value of from about 90 to about 110. A tenderometer is a device that is used to measure the tenderness of peas, beans and broad beans. As used herein, the term “tenderometer value” refers to a measure of the force required to crush about 200 grams of peas using a tenderometer. Higher tenderometer values indicates more advanced maturity of the peas. Processed green peas are typically harvested at tenderometer values of from 90 to 120.

The peas of the present invention contain from about 5 to about 30 percent fresh weight, preferably from about 10 to about 20 percent fresh weight, more sucrose than peas produced from a Pisum sativum variety that does not contain the bsg gene homozygous within its genome, particularly wrinkled varieties of peas. As used herein, the term “wrinkled varieties of peas” refers to Pisum sativum that contain a homozygous r (rugosus) or homozygous rb gene within their genome, so that the appearance of the mature seed is wrinkled. Quantum is the sweetest variety of pea that is known by the inventor that contains the r gene homozygous within its genome. Quantum is commercially available under the Asgrow Brand, from Seminis Vegetable Seeds, Inc., the assignee of the present invention. Quantum peas contain from about 5.0 to about 6.2 percent fresh weight of sucrose.

Additionally, the peas of the present invention exhibit a depressed level of alcohol insoluble solids (AIS). Low AIS correlates with high product quality. The peas of the present invention contain from about 6.5 to about 8.0, preferably from about 7.0 to about 7.5 percent by weight of alcohol insoluble solids when measured at a tenderometer value of about 105. Moreover, the peas of the present invention exhibit about twenty (20) percent less AIS than peas from a Pisum sativum variety that does not contain the bsg gene homozygous within its genome (such as wrinkled varieties of peas). For example, peas of the present invention contain about twenty (20) percent less AIS than the peas produced by the variety Quantum.

Furthermore, the mature seeds of the present invention contain very little starch. More specifically, the seeds of the present invention contain from about 0.01 to 3.0 percent dry weight, preferably, from about 0.5 to 2.0 percent dry weight of starch.

Breeding History

The Pisum sativum variety of the present invention containing a homozygous bsg gene within its genome was developed as follows. In 1990, a wrinkled-seed mutant was discovered in a crop of Frimousse, which is a smooth-seed Pisum sativum variety that has no block in the conversion of sugar to starch (this mutant is designated “Frimousse introgressed-bsg”). This crop had been grown at a breeding station in Twin Falls, Id. This single mutant was discovered when the crop was undergoing seed-uniformity tests. Frimousse is commercially available under the Asgrow Brand, from Seminis Vegetable Seeds, Inc. A plant of this wrinkle-seed mutant was then crossed with the Pisum sativum variety Encore in a greenhouse in Twin Falls, Id. Encore is a full season, large sieve commercial Pisum sativum variety with dark green (freezer) berry color, good processed quality, and resistance to Powdery Mildew Fungus and Race 1 of the Fusarium Wilt Fungus. Dry, mature seed of Encore is available under the Asgrow Brand from Seminis Vegetable Seeds. One selection that resulted from this cross contained very little starch and was labeled M82.1.

In December 1991, seed of M82.1 and the variety Lazor were planted in a greenhouse in Twin Falls, Id. A cross was made between M82.1 and Lazor. Lazor is a late-season, large sieve Pisum sativum variety with dark green berry color and good processed quality and is resistant to Powdery Mildew Fungus and Race 1 of Fusarium Wilt Fungus. Dry, mature seed of Lazor is commercially available under the Asgrow brand from Seminis Vegetable Seeds, Inc., the assignee of the present invention. The resulting mature seed was collected and coded “BC0”. In May 1992, seed of BC0 and Lazor were planted in the greenhouse described above and crossed. The resulting mature seed was collected and coded “BC1”. In September 1992, seed of BC1 and Lazor were planted in the greenhouse described above and crossed. The resulting mature seed was collected and coded “BC2F1”. In February 1993, seed of BC2F1 were planted in the same greenhouse and allowed to self-pollinate. The resulting mature seeds were collected and coded “BC2F2”. In April 1993, several BC2F2 seeds that did not contain any organized starch grains were selected.

Later in 1993, the BC2F2 seeds selected above were then planted in a field in Twin Falls, Id. From the resulting population, individual lines containing the best horticultural characteristics were then selected and allowed to self-pollinate. Selection criteria included uniform emergence, short erect plant type, high productivity and uniformly of pod set. The resulting mature seeds were collected and coded “BC2F3”. In April 1994, BC2F3 seed was planted in Twin Falls, Id.

From the resulting population, individual lines exhibiting the best horticultural characteristics were selected and allowed to self-pollinate. The resulting seed was collected and coded “BC2F4”. In April 1995, BC2F4 seed was planted under the code name “R954593” in Twin Falls, Id. Observations made during the growing season indicated that the line was uniform and bred true. The resulting variety was allowed to self-pollinate and the resulting seed was collected and coded 8500017. In winter 1995, the 8500017 seed was planted in Guatemala and the resulting seed bulked for further trialing and increase.

Mature seeds of 8500017 developed as a result of the above breeding have been deposited under the Budapest Treaty with the American Type Culture Collection (hereinafter “ATCC”), 12301 Parklawn Drive, Rockville, Md., 20852 on Nov. 3, 1997 and have received accession number 209425. This deposit of 8500017 was made under the Budapest Treaty and will be maintained in the ATCC depository, which is a public depository, for a period of 30 years, or 5 years after the most recent request, or for the enforceable life of the patent, whichever is longer, and will be replaced if it becomes nonviable during that period. Additionally, Applicant has satisfied all the requirements of 37 C.F.R. Sections 1.801-1.809, including providing an indication of the viability of the sample. Furthermore, a Plant Variety Protection Certificate has been applied for on Feb. 19, 1998 with the United States Department of Agriculture and assigned PVP Application Number 9800121.

Processes for Developing Pisum sativum Varieties Containing Higher Levels of Sucrose

The present invention also contemplates a process for developing Pisum sativum varieties that produce peas that contain higher levels of sucrose than peas produced by a Pisum sativum variety that does not contain the bsg gene within its genome. In one embodiment, the process involves crossing a Pisum sativum variety or line that contains the bsg gene homozygous within its genome with a second Pisum sativum variety or line that also contains the bsg gene homozygous within its genome, collecting the resulting mature seed, planting the mature seed, growing the mature seed into Pisum sativum plants, selecting Pisum sativum plants with desirable phenotypic traits, allowing the selected plants to self-pollinate until a uniform line is produced, allowing the Pisum sativum line to self-pollinate, and collecting the resulting peas.

In another embodiment, the process involves crossing a Pisum sativum variety or line that contains the bsg gene homozygous within its genome with a second Pisum sativum variety or line that does not contain the bsg gene within its genome and collecting the resulting mature seed. The Pisum sativum variety or line that does not contain the bsg gene within its genome can instead contain other genes or combination of genes within its genome. For example, the variety or line may contain the r or rb gene, or both, which produces wrinkled seed, or the R gene which produces smooth seed. The collected mature seed contains the bsg gene heterozygous within its genome. The collected mature seed are planted and allowed to grow. The Pisum sativum plants grown from said seed are allowed to self-pollinate. As a result of the self-pollination, the plants produce various types of dry, mature seeds that have different genes within their genomes. One type of mature seed that is produced is highly wrinkled. This seed contains virtually no organized starch grains and does not stain purple when treated with a solution of iodine and potassium iodine solution. This mature seed contains the bsg gene homozygous within its genome. The mature seed containing the bsg gene is selected, collected and planted. Pisum sativum plants having desirable phenotypic traits are selected and allowed to self-pollinate until a uniform Pisum sativum line is produced. The uniform line is allowed to self-pollinate and the resulting peas collected.

As used herein, the term “line” means a population of genetically and phenotypically similar seeds or plants.

As used herein, the term “uniform Pisum sativum line” means a population of genetically and phenotypically similar seeds or plants that may be reproduced by bulk increase without altering the descriptive characteristics of the population. A variety is a uniform that is increased for commerce.

The present invention also contemplates peas produced by any of the hereinbefore described processes. Peas produced pursuant to the above-mentioned processes contain from about 6.0 to about 7.5, preferably from about 6.5 to about 7.0 percent fresh weight sucrose when measured at a tenderometer value of from about 90 to about 110 and from about 6.5 to about 8.0, preferably from about 7.0 to about 7.5 percent by weight alcohol insoluble solids when measured at a tenderometer value of about 105. Moreover, peas produced by the process of the present invention also contain higher levels of sucrose than peas produced from a Pisum sativum variety that does not contain the bsg gene homozygous within its genome. Moreover, peas produced according to the process of the present invention contain from about 5 to about 30 percent fresh weight, preferably from about 10 to about 20 percent fresh weight, more sucrose than peas produced by a Pisum sativum variety that does not contain the bsg gene within its genome. Additionally, peas produced according to the process of the present invention contain about twenty (20) percent less AIS than peas produced from a Pisum sativum variety that does not contain the bsg gene within its genome.

The present invention also contemplates a process for producing highly wrinkled mature seed of a Pisum sativum variety that contains the bsg gene homozygous within its genome. In one embodiment, the process involves crossing a Pisum sativum variety or line that contains the bsg gene homozygous within its genome with a second Pisum sativum variety or line that contains the bsg gene homozygous within its genome and collecting the resulting mature seeds. The present invention also contemplates Pisum sativum varieties grown from said dry, mature seed and peas harvested from said varieties.

In a second embodiment, the process involves crossing a Pisum sativum variety or line that contains the bsg gene homozygous within its genome with a second Pisum sativum variety or line that does not contains the bsg gene homozygous within its genome and collecting the resulting mature seeds. After the mature seed is collected, it is planted and grown into plants which are allowed to self-pollinate. The resulting mature seed is collected and highly wrinkled mature seeds that do not contain any organized starch grains are selected. These mature seeds are planted and allowed to grow into Pisum sativum plants. Pisum sativum plants having desirable phenotypic traits are selected. These selected plants are allowed to self-pollinate until a uniform Pisum sativum line is produced. The uniform line is allowed to self-pollinate and mature seeds collected. The present invention also contemplates Pisum sativum varieties grown from said dry, mature seed and peas harvested from said varieties.

Molecular Characterization of the bsg Gene

Pisum sativum varieties containing the bsg gene have been found to exhibit a significant reduction in the activity of the enzyme plastid phosphoglucomutase (hereinafter referred to as “PGM”) in leaves and cotyledons (see FIGS. 4 and 5). The significance of this lack of plastid PGM activity is that in the plastid, the conversion of glucose-6-phosphate to glucose-1-phosphate cannot occur. The importance of this reaction in the synthesis of starch is that glucose-1-phosphate is the substrate for the committed pathway of starch synthesis. Without a supply of glucose-1-phosphate, the synthesis of starch cannot take place. Sugar and starch metabolism are known to be related in plants. It is believed that an alteration in the levels of an enzyme involved in the starch synthesis pathway results in an alteration of the level of sugar in a plant and thus higher levels of sucrose in the seed (peas).

It has been discovered that Pisum sativum varieties containing the bsg gene contain a mutation in a 3′ splice site in an intron in a gene encoding the plastid phosphoglucomutase. More specifically, the mutation is in the 3′ splice site dinucleotide AG, where nucleotide A is replaced with nucleotide T.

Transcription is the synthesis of RNA on a DNA template by complementary base pairing within the nucleus of a cell. The RNA molecule initially transcribed from the DNA template is known as the “primary transcript”. The primary transcript which is transcribed from the DNA contains “introns”, which are frequently referred to as “intervening sequences.” Introns are absent from the mature form of the primary transcript which is referred to as “messenger RNA” or “mRNA” in short. Introns are removed during post-transcriptional modification of the primary transcript. A number of events take place during the post-transcriptional modification of the primary transcript, one of which includes the removal of the introns and the splicing together of exons during a number of reactions which occur within a spliceosome. Once the post-transcriptional modifications are completed, a mature mRNA leaves the nucleus and enters the cytoplasm.

In the present invention, the mutation in the 3′ splice site in an intron in a gene encoding the plastid phosphoglucomutase prevents the excision of this intron during post-transcriptional modification of the primary transcript. Because the intron is not excised from the primary transcript, it gives rise to three aberrant mRNAs. As used herein, the term “aberrant” means different from the correctly spliced wildtype plastid PGM mRNA by containing unexcised intron sequences or incorrectly excised exons. When translated, the aberrant mRNAs produces a plastid PGM polypeptide that is truncated and hence, shorter in length than the polypeptide produced by the wildtype, which is about 627 amino acids in length (See WO 98/01574 which reports the cloning of a polynucleotide sequence encoding the pea plastidical phosphoglucomutase wildtype having an amino acid sequence of about 627 amino acids). The alteration of this plastid PGM polypeptide is believed to be responsible for the significant reduction in activity of this enzyme in Pisum sativum varieties containing the bsg gene. This mutation of the bsg gene shall be more fully described below.

A portion of the plastid PGM genomic nucleotide sequence of the bsg mutant has been characterized and is shown in FIG. 6 and in SEQ ID NO:1. A portion of the plastid PGM genomic nucleotide sequence from a conventional, smooth-seed Pisum sativum variety, called “Frimousse”, which is commercially available from Seminis Vegetable Seeds, Inc., the assignee of the present invention, has been characterized and is shown in FIG. 7 and SEQ ID NO:2. This conventional, smooth-seed Pisum sativum variety shall hereinafter be referred to as the “wildtype”. In FIGS. 6 and 7, exons are shown in bold-faced type.

A comparison of the genomic nucleotide sequence of the wildtype with the genomic nucleotide sequence of the bsg mutant revealed a single mutation in one of the introns in the nucleotide sequence of plastid PGM in the bsg mutant. Specifically, in the bsg mutant, at nucleotide 1594, an A in the 3′ splice site dinucleotide AG/ is replaced with a T (See asterisk in FIGS. 6 and 7). Splice sites are regions containing a few nucleotides that reside at the end of introns and function in excision and splicing reactions during the processing of transcripts from genes. The 3′ splice site dinucleotide AG/ is invariant in naturally occurring higher plant introns (Simpson and Filipowicz Plant Mol. Biol. 32:1-41 (1996). It is known in maize, for example, that this same mutation, AG'TG, abolishes processing (see Carle-Urioste et al. Plant Mol. Biol. 26:1785-1795 (1994)).

This mutation in the 3′ splice site results in three aberrant transcripts being produced during transcription. The largest transcript contains not only the intron with the mutated 3′ splice site but a downstream intron as well. It is not clear why this downstream intron is retained. The retention of these two introns in this transcript results in a plastid PGM polypeptide which is immediately truncated by a termination codon near the 5′ end of the first retained intron. This results in the loss of about 173 amino acids from the carboxyl terminal of the plastid PGM polypeptide. Northern analysis suggests that this first transcript is the major transcript which accumulates in the bsg mutant (see FIG. 8).

The second transcript is approximately the same size as the wildtype transcript, but is missing about the first 11 nucleotides of the exon directly downstream from the mutated intron. This results from the utilization of a cryptic AG 3′ splice site dinucleotide in the downstream exon. As a result, a frame shift mutation is created and 23 amino acids are substituted from the wrong reading frame for the C-terminal 174 amino acid of plastid PGM.

The third transcript is smaller than the wildtype transcript due to a deletion of the 142 base pair exon immediately downstream from the mutated intron. This results from the substitution of the 3′ splice site in the downstream intron for the mutated 3′ spice site. As a result, a frameshift mutation is created and 9 amino acids are substituted for the C-terminal 173 amino acids of plastid PGM.

The modified plastid PGM polypeptide encoded by the nucleotide sequence of SEQ ID NO:1 and shown in FIG. 6 is believed to be responsible for the significant reduction in the activity of this enzyme in varieties containing this mutation.

It should be recognized that Pisum sativum varieties containing mutations in the plastid PGM gene in addition to the mutation described above in at the 3′ splice site in the intron of the plastid PGM gene, as well as mutations in genes other than the plastid PGM gene, are also encompassed by this invention.

Moreover, it should further be recognized that the present invention encompasses any plant which contains one or more mutations in a gene encoding plastid phosphoglucomutase. Specifically, the gene must contain at least one mutation in one or more introns. The mutation must prevent the excision of one or more introns from a primary transcript during post-transcriptional modification of the transcript which results in the production of one or more aberrant mRNA molecules for translation. During translation, this aberrant mRNA produces a plastid PGM polypeptide which is either truncated or larger than the wildtype plastid PGM polypeptide. The types of plants encompassed by the present invention include “monocotyledonous” (often referred to as “monocots”) or “dicotyledonous” (often referred to as “dicots”) plants. A “monocotyledonous plant” refers to a plant whose seeds have only one cotyledon, or organ of the embryo that stores and absorbs food. A “dicotyledonous plant” refers to a plant whose seeds have two cotyledons.

DNA Molecules of the Present Invention

In another embodiment, the present invention relates to a DNA molecule. It is believed that the introduction into a plant of an expression vector containing an isolated and purified DNA molecule of the present invention (as defined hereinafter) inserted in either the sense or antisense direction will result in an alteration, specifically, a reduction, in the levels of expression of the native plastid PGM gene. For example, it is well known that gene expression in transgenic plants can be inhibited through the use of sense RNA transcribed from an exogenous template to downregulate the expression of specific plant genes (see Jorgensen, Keystone Symposium “Improved Crop and Plant Products through Biotechnology”, Abstract X1-022 (1994)). The use of sense suppression is well known in the art and is described in Waterhouse, P M., et al., Proc. Natl. Acad. Sci. U.S.A., 95(23):13959-64 (1998), Oue Q, et al., Plant J. 13(3):401-9 (1998) and Tsai C J., et al, Plant Physiol. 117(1):101-12 (1998), all incorporated herein by reference. It is well known, a cell manufactures protein by transcribing the DNA of the gene encoding that protein to produce RNA, which is then processed to messenger RNA (hereinafter referred to as “mRNA”) and finally translated by ribosomes into protein. This process may be inhibited in the cell by the presence of antisense RNA. The term “antisense RNA” means an RNA sequence which is complementary to a sequence of bases in the mRNA in question in the sense that each base or them majority of bases in the antisense sequence (read in the 3′ to 5′ sense) is capable of pairing with the corresponding base (G with C, A with U) in the mRNA sequence read in the 5′ to 3′ sense. It is believed that this inhibition takes place by formation of a complex between the two complementary strands of RNA, thus preventing the formation of protein. How this works is uncertain: the complex may interfere with further transcription, processing, transport or translation, or degrade the mRNA, or have more than one of these effects. This antisense RNA may be produced in the cell by transformation of the cell with an appropriate DNA construct arranged to transcribe the non-template strand (as opposed to the template strand) of the relevant gene (or of a DNA sequence showing substantial homology therewith).

The use of antisense RNA to downregulate the expression of specific plant genes is well known. Reduction of gene expression has led to a change in the phenotype of the plant: either at the level of gross visible phenotypic difference, e.g., lack of anthocyanin production in flower petals of petunia leading to colorless instead of colored petals (see van der Krol et al., Nature, 333:866-869 (1988)); or at a more subtle biochemical level, for example, a change in the amount of polygalacturonase and reduction in depolymerization of pectin during tomato fruit ripening (see Smith et al., Nature, 334:724-726 (1988)).

Additionally, the DNA molecule of the present invention can be used in site-specific (also known as “homologous” and “gene targeting”) recombination. It is known for example, that the presence of isogenic genomic sequences on an introduced DNA can be targeted to its own chromosomal locus and thus facilitate integration by site-specific recombination (Schaefer, D. G., et al., Plant J., 11(6):1195-1206 (1997), herein incorporated by reference). Initially observed in Saccharomyces cerevisiae, this event is a prerequisite to application on the most sophisticated tools of reverse genetics, i.e. gene disruption and allele replaced. Id. With such approaches, virtually any cloned gene, even of unknown function, can be specifically mutagenized in vitro and re-introduced to its own chromosomal location in order to study its function. Id. The successful application of allele replacement depends on the ratio of homologous to illegitimate recombination events during integrative transformation. Id.

The DNA molecule of the present invention can be used in site-specific recombination to replace the wildtype nucleotide sequence to give rise to non-functional plastid PGM activity. Preferably, the nucleotide sequence of the DNA molecule used in such site-specific recombination exhibits a high homology with the target wildtype nucleotide sequence being replaced and contains about 50 to about 10,000 nucleotides, most preferably about 5,000 nucleotides. The techniques for site-specific recombination described in Risseeuw et al., Plant J., 7(1):109-19 (1995), Offringa R., et al., Proc. Natl. Acad. Sci. U.S.A. 90(15):7346-50 (1993), Offringa R., et al., EMBO J., 9(10):3077-84 (1990), Miao, Z H Plant J., 7(2):359-65 (1995), all herein incorporated by reference, can be used to effect the site-specific recombination in the present invention.

The DNA molecule of the present invention comprises: (1) a nucleotide sequence comprising the nucleotide sequence shown in SEQ ID NO:1 which contains a mutation in an intron at nucleotide 1594 at the 3′ splice site where an A is replaced by a T and sequences which are complementary to the sequence shown in SEQ ID NO:1; and (2) a nucleotide sequence encoding plastid phosphoglucomutase, where the nucleotide sequence contains at least one nucleotide which prevents the excision of at least one intron from a primary transcript during post-transcriptional modification of the transcript and which produces at least one aberrant mRNA for translation. The nucleotide sequences described herein can be isolated and purified from a natural substance such as a plant or can be artificially synthesized and then purified using techniques which are well known in the art. Additionally, the present invention also relates to an expression vector containing this DNA molecule, plant cells transformed with these vectors, and a process of reducing or suppressing plant plastid PGM expression by sense or antisense suppression in plants transformed with these vectors.

In another aspect, the present invention also contemplates naturally occurring allelic variations and mutations of the nucleotide sequences set forth above so long as the DNA molecule contains the hereinbefore described mutation.

In yet another aspect, the present invention also includes nucleotide sequences which hybridize under stringent hybridization conditions to the nucleotide sequences set forth above. Stringent hybridization conditions are well known in the art and define a degree of sequence identity greater than 70% to 80%.

The present invention also contemplates oligonucleotides from about 15 to about 50 nucleotides in length, which serve as primers and hybridization probes for the screening of DNA libraries and the identification of DNA or RNA molecules containing the mutation described hereinbefore and related sequences. Such primers and probes are characterized in that they will hybridize to the DNA molecule containing the hereinbefore described mutation contained within said DNA molecule. An oligonucleotide probe or primer contains a nucleotide sequence of at least 15 nucleotides that is identical to, nearly identical to or complementary to a contiguous sequence the DNA molecule shown in SEQ ID NO:1. Thus, where an oligonucleotide probe is 25 nucleotides in length, at least 15 of those nucleotides are identical or complementary to a sequence of the DNA molecule of the present invention.

In yet another aspect, the present invention also provides DNA constructs comprising all or part of the DNA molecule described above. A “construct” as used herein, is a DNA molecule comprising nucleic acid sequences not normally associated in nature, such as a prokaryotic sequence and a eukaryotic sequence. Typically, a “construct” comprises a vector, such as a plasmid, viral, and/or episomal origin, and a sequence to be transcribed.

Generally, the DNA construct will contain at least one promoter. The promoters may be heterologous, meaning that they are not naturally operably linked to a plastid PGM gene. Promoters useful for expression in plants are well known in the art and can be inducible, constitutive, tissue specific, derived from eukaryotes, procaryotes or viruses, or have various combinations of these characteristics. Examples of promoters that are suitable for use in plants include the cauliflower mosaic virus 35S promoter, the phytohemagglutinin (PHA) promoter, ribulose-1,5-bisphosphate carboxylase (rbcs) promoters and chlorophyll a/b binding protein (Cab) promoters.

Selection of an appropriate expression vector is relatively simple, as the constraints are minimal. The minimal traits of the vector are that the desired nucleotide sequence be introduced in a relatively intact state. For example, if plant cells are to be transformed with the DNA construct, any vector that will produce a plant carrying the introduced DNA sequence should be sufficient.

Thus, suitable vectors include the Ti plasmid vectors, shuttle vectors designed merely to maximally yield high numbers of copies, episomal vectors containing minimal sequences necessary for ultimate replication once transformation has occurred, transposon vectors, homologous recombination vectors, mini-chromosome vectors, and viral vectors. The selection of vectors and methods to construct them are commonly known to persons of ordinary skill in the art and are described in general technical references, such as Sambrook et al., (1989) Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Vols. 1-3, which is incorporated herein by reference.

The vector may also include any additional attached nucleotide sequences which will confer resistance to the degradation of the DNA molecule to be introduced, which assists in the process of genomic integration or which provides a means to easily select for transformed cells or plants are advantageous and greatly decrease the difficulty of selecting useable transgenotes. Commonly, expression vectors will contain selection markers, such as kanamycin resistance, hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired DNA sequences (see U.S. Pat. No. 4,704,362, which is herein incorporated by reference).

Useful vectors will generally contain sequences that allow replication in a prokaryotic host useful for cloning the DNA molecule of the present invention. The most commonly used prokaryotic hosts are strains of Escherichia coli, although other prokaryotes, such as Bacillus subtilis or Pseudomonas may also be used, and are well known in the art. Useful vectors may also contain other sequence elements useful for cloning (for example, restriction sites) or expression (for example, enhancer sequences).

Useful expression vectors are well known in the art and are readily available. Typically, the vector will contain a polyadenylation signal, such as the polyadenylation signal from the cauliflower mosaic 35S gene. The vector may also contain translation regulatory sequences (such as translation start sites), and may also contain introns and splice sites, enhancer sequences (which can be inducible, tissue specific or constitutive), and 5′ and 3′ regulatory and flanking sequences.

To achieve expression, it is necessary to introduce the appropriate construct into at least some cells of a host organism, such as by transformation. As used herein, the term “transformation” means alteration of the genotype (including episomal genes) of a target organism by the introduction of a nucleic acid sequence. The nucleic acid sequence need not necessarily originate from a different source, but it will, at some point, have been external to the cell into which it is to be introduced.

The host organism can be yeast cells, such as Saccharomyces cerevisiae, plant cells such as Pisum sativum cells, insect cells such as Tn5 cells and bacterial cells such as E. coli and Pseudomanas.

The transformation of plants may be carried out in essentially any of the various ways known to those skilled in the art of plant molecular biology. See, in general, Methods in Enzymology Vol. 153 (“Recombinant DNA Part D”) 1987, Wu and Grossman Eds., Academic Press, incorporated herein by reference. As used herein, the term “plant” refers to whole plants and plant-derived tissues. As used herein, “plant-derived tissues” refers to differentiated and undifferentiated tissues of plants, including, but not limited to roots, shoots, leaves, pollen, ovules, seeds, tumor tissue, and various forms of cells in culture such as intact cells, protoplasts, embryos and callus tissue. Plant-derived tissues may be in planta or in organ, tissue or cell culture. A “monocotyledonous plant” refers to a plant whose seeds have only one cotyledon, or organ of the embryo that stores and absorbs food. A “dicotyledonous plant” refers to a plant whose seeds have two cotyledons. A “protoplast” refers to a plant cell without a cell wall or extracellular matrix.

The vector may be introduced into plant cells by microinjection, by using polyethylene glycol (Paszkowski et al. (1984) EMBO J. 3:2717-2722), by electroporation (Fromm et al. (1985) Proc. Natl. Acad. Sci. USA 82:5824-5828), by high ballistic penetration by small particles with the DNA molecule either within the matrix of small beads or particles, or on the surface (Klein et al., (1987) Nature 327:70-73).

A preferred method of introducing the vector into plant cells is to infect a plant cell, an explant, a meristem or a seed with a genetically engineered Agrobacterium tumefaciens or Agrobacterium rhizogenes strain carrying the segment. Within the T-DNA segment of its full-size Ti plasmid or on an abbreviated binary Ti plasmid vector containing the T-DNA boundary sequences of the Agrobacterium tumafaciens Ti plasmid is, if used, must be “disarmed”, i.e., have its tumor-inducing activity removed, prior to use. To facilitate selection of transgenic plant cells, it is preferable that the gene segment be linked to a selectable marker, for example, kanamycin resistance. In some species, such as Arabidopsis thaliana, this Agrobacterium infection process is facilitated by vacuum infiltration of embryonic tissue (as in Becktold et al., (1993) C.R. Acad. Sci. Paris 316:1194-1199). Examples of Agrobacterium tumefaciens strains that can be used include LBA4404, as described by Hoekema et al., (1983) Nature 303:179-180, and EHA101 as described by Hoot et al., (1986) J. Bacteriol. 168:1297-1301. A preferred Agrobacterium rhizogenes strain is 15834, as described by Birot et al., (1987) Plant Physiol. Biochem. 25:323-325. The Ti plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and is stably integrated into the plant genome (Horsch et al., (1984) Science 233:496-498; Fraley et al., (1983) Proc. Natl. Acad. Sci. USA 80:4803-4807). Under appropriate conditions known in the art, the transformed plant cells are placed under antibiotic selection and grown in tissue culture media to form culture shoots, roots, and eventually intact plants which can be propagated in soil.

Ti plasmids contain two regions essential for the production of transformed cells. One of these, named transfer DNA (T-DNA), induces tumor formation. The other, termed virulent region, is essential for the introduction of the T-DNA into plants. The transfer DNA region, which transfers to the plant genome, can be increased in size by the insertion of the foreign nucleic acid sequence without its transferring ability be affected. By removing the tumor-causing genes so that they no longer interfere, the modified Ti plasmid an then be used as a vector for the transfer of the gene constructs of the invention into an appropriate plant cell, such being a “disabled Ti vector”.

There are presently at least three different ways to transform plant cells with Agrobacterium:

(1) co-cultivation of Agrobacterium with cultured isolated protoplasts;

(2) transformation of cells or tissues with Agrobacterium; or

(3) transformation of seeds, apices, meristems or whole plants with Agrobacterium.

Method (1) requires an established culture system that allows plant regeneration from cultured protoplasts. Method (2) requires (a) that the plant cells or tissues can be transformed by Agrobacterium and (b) that the transformed cells or tissues can be induced to regenerate into whole plants. Method (3) requires regeneration or micropropagation or simply “propagation” of Arabidopsis seeds transformed with a vector.

In the binary system, to have infection, two plasmids are needed: a T-DNA containing plasmid and a vir plasmid. Any one of a number of T-DNA containing plasmids can be used, the only requirement is that one be able to select independently for each of the two plasmids.

As a result of this plant cell transformation process, a Ti plasmid segment carrying the desired DNA segment is integrated in the nuclear chromosome and transformed cells can be selected by using a selectable marker linked to the desired DNA segment. These selectable markers include, but are not limited to, antibiotic resistance, herbicide resistance or visually-assayable activities. Other selectable markers known in the art may be used in this invention.

Normally, regeneration will be involved in obtaining a complete, transgenic organism from the transformation process. For example, in plants, the term “transgenote” refers to the immediate product of the transformation process and to resultant whole transgenic plants. The term “regeneration” as used herein, means growing a whole or complete transgenic organism. For example, in plants, the term regeneration relates to growing a whole plant form a plant cell, a group of plant cells, a plant part, a plant piece (e.g., from a protoplast, callus, or tissue part), or the propagation of seeds transformed with Agrobacterium by vacuum infiltration.

In plants, regeneration from cultured protoplasts is described in Evans et al., Protoplast Isolation and Culture in Handbook of Plant Cell Cultures 1:124-176 (MacMillan Publishing Co. New York 1983); M. R. Kavey, “Recent Developments in the Culture and Regeneration of Plant Protoplasts,” Protoplasts (1983)-Lecture Proceedings, pp. 12-29, (Birkhauser, Basal 1983); P. J. Dale, “Protoplasts Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops”, Protoplasts (1983)-Lecture Proceedings, pp.31-41 (Birkhauser, Basel (1983); and H. Binding, “Regeneration of Plants,” Plant protoplasts pp. 21-73 (CRC Press, Boca Raton 1985).

Identification, selection or confirmation of transgenic organisms is typically based on an assay or assays. Transgenic organisms (such as transgenotes) can be screened by biochemical, molecular biological, and other assays. For example, various assays may be used to determine whether a particular plant, plant part, or transgenote cell shows an increase (i.e., overexpression) or reduction (i.e., suppression) of the plastid PGM gene. Typically the change in expression or activity of the transgenote will be compared to levels found in wildtype (e.g., untransformed) plants of the same type. Preferably, the effect of the introduced construct (transgene) on the level of expression or activity of the endogenous gene will be established from a comparison of sibling plants with and without the construct containing the desired DNA fragment. mRNA levels can be measured by Northern blotting, primer extension, ribonuclease protection, quantitative or semi-quantitative PCR (polymerase chain reaction), and other methods well known in the art (see, e.g., Sambrook et al., (1989)). Protein can be measured in a number of ways including immunological methods such as by ELISA or Western blotting.

The following Examples illustrate the preferred embodiments of the present invention and are not limiting of the specification and claims in any way.

EXAMPLE 1 Characteristics of 8500017

Pea cultivar 8500017 has the following characteristics:

Maturity:

Number of Nodes at first bloom: 15

The variety of the present invention blooms 5 days later than Wando. Wando is publically available from the Pisum Plant Introduction Collection, USDA ARF, Johnson Hall, Room 55, Washington State University, Pullman, Wash.

Number of days to procuring: 77

Host Units: 1630

Height: The variety of the present invention is about 45 cm high, and is about 5 cm shorter than Wando.

Vine:

Habit: Indeterminate

Branching: 1-2 Branches

Internodes: Zig Zag

Stockiness: Slim

Number of Nodes: 19

Leaflets:

Color: Dark Green

Wax: Light

Marbled: Yes

Number of leaflet pairs: Two

Stipules: The variety of the present invention contains stipules that are marbled. The stipules are the same color as the leaflets, are clasping and are larger in size when compared to the leaflets.

Flower color:

Venation: Greenish

Standard: White

Wing: White

Keel: White

Pods:

Shape: Slightly Curved

Color: Medium Green

Surface: Rough

Length: 8 cm

End: Blunt

Width (Between Sutures): 11 mm

Number of Seeds per Pod: 9

Peas: (95-100 Tenderometer)

Color: Dark Green

Sieve:

1—5%

2—17%

3—35%

4—39%

5—4%

Average: 3.20%

Seeds: (Dry, Mature):

Shape: Flattened

Surface: Wrinkled and Dull

Color Pattern: Monocolor

Primary Color: Cream & Green

Hilum Floor Color: Tan

Cotyledon Color: Green

Number of grams per 100 Seeds: 14

Disease Resistance: The Pisum sativum variety of the present invention is resistant to Fusarium Wilt Fungus and Powdery Mildew Fungus

The closest Pisum sativum varieties to the Pisum sativum variety of the present invention are: Lazor, Markado and Trek. Lazor has been described earlier. Markado is a full season, medium sieve, freezer with dark green (freezer) peas and with relatively fine foliage compared to other larger sieve freezer varieties. Markado is commercially available from Novartis (Boise, Id.). Trek is a mid-season, medium-sieve freezer variety with afila foliage and medium dark green berry color. Trek is commercially available under the Asgrow Brand from Seminis Vegetable Seeds.

EXAMPLE 2 Starch and Sucrose in the Leaves of the Peas Present Invention and Examination of Enzyme Activity Compared with Peas of a Wrinkled Pisum sativum Variety that Contains the r Gene

The pea variety Lazor, which contains homozygous r gene and produces wrinkled mature seed, and a bsg-introgressed line of Lazor which produces highly wrinkled seed were studied in this example. A sample of leaves from Lazor and bsg-introgressed line of Lazor were dried to a stable weight in a lyophilizer and then pulverized to a powder using a mortar and pestle. Pre-weighed amount of the samples (˜100 mg) was washed several times in 80% ethanol at 70° C. The ethanol supernatants were pooled and used for sucrose determination. The pellet was used for starch determination.

Quantification of Starch

Sugar was removed from lyophilized tissue samples by several washes at 70° C. in 80% ethanol. Starch was gelatinized by autoclaving for 45 minutes and then hydrolyzed by digestion at 37° C. with α-amylase (Sigma) in 0.3 M sodium acetate, pH 5.2 followed by further digestion with Aspergillus niger amyloglucosidase (Sigma) in 0.1 M sodium acetate, pH 4.6 at 55° C. Samples were deproteinized by boiling and glucose was assayed enzymatically by coupling the oxidation of glucose to the reduction of NAD+ with hexokinase (Boehringer/Mannheim) and glucose-6-P dehydrogenase (Sigma).

An aliquot of the hydrolyzed starch was incubated at room temperature in 0.1 M Tris-Cl (pH 8), 1 mM ATP, 1 mM NAD+, 2 mM MgCl₂, 15 U/ml yeast hexokinase, and 15 U/ml Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase. The reaction was allowed to go to completion and the absorbance was read at 340 nm and compared with a standard curve. The results are shown below in Table 1.

Quantification of Sucrose

The pooled 80% ethanol supernatants were brought to 6.5 mls with 80% ethanol. Following the addition of 3.6 ml of water and 2.15 ml of chloroform, the sample was vortexed and spun in a table top centrifuge. After the bottom phase had been back-extracted with 0.2 ml of water, the top phases were pooled, evaporated to dryness on a Savant Speed-Vac Concentrator, and resuspended in 0.2 ml of water.

Glucose and fructose were destroyed by treating the sample for 30 minutes at 90° C. in the presence of 3.7 volumes of 0.1 M NaOH. The sample was then neutralized by the addition of 0.5 volumes of 1 M sodium acetate, pH 4.6. Sucrose levels were determined using a dehydrogenase-coupled spectrophotometric assay. An aliquot of the alkali-treated sample was incubated at room temperature in 40 mM imidazole (pH 6.9), 1 mM ATP, 1 mM NAD+, 5 mM MgCl₂, 0.5 mM DTT, 0.02% BSA, 32 U/ml invertase, 5 U/ml yeast hexokinase, and 5 U/ml Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase. The reaction was allowed to go to completion and the absorbance was read at 340 nm and compared with a standard curve. The results are shown below in Table 1.

TABLE 1 Starch and Sucrose Levels in Leaves from Lazor and bsg -introgressed lines of Pisum sativum Starch Starch Sucrose Sucrose % dry wt % fresh wt % dry wt % fresh wt Lazor 3.060 +/− 0.4948 +/− 8.18 +/− 0.69 1.32 +/− 0.14 0.071 0.0220 bsg 0.015 +/− 0.0023 +/− 10.19 +/− 0.78  1.58 +/− 0.12 0.002 0.0004

Table 1 shows that leaf starch levels in the Pisum sativum line having the bsg gene introgressed into Lazor are at most only about 0.5% of Lazor. Also, Table 1 demonstrates that sucrose levels were increased in the leaves of the Pisum sativum line containing the bsg gene.

ADP-glucose Pyrophosphorylase Assays

Embryos from Lazor and a bsg gene introgressed line of Lazor frozen in liquid nitrogen were ground in a mortar in the presence of 3-4 volumes of ice-cold extraction buffer (100 mM 2-(N-morpholino) ethane sulfonic acid (MOPS) (pH 7.2), 5 mM MgCl₂, 1 mM EDTA, 5 mM DTT). Starch and cell debris were removed by pelleting for 10 minutes at 27,000×g. Aliquots of the supernatant were then quick-frozen in liquid nitrogen and stored at −80° C. ADPG-PPase activity was found to be stable under these storage conditions.

ADP-glucose pyrophosphorylase was assayed in the reverse direction at 25° C. by coupling production of glucose-1-P to reduction of NAD+. Standard reaction mixtures (1 ml) contained 75 mM 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid (HEPES) (pH 7.75), 5 mM MgCl₂, 1 mM 3PGA, 0.1 mg/ml bovine serum albumin, 5 μM glucose-1,6-bisphosphate; 1.5 mM ADP-glucose, 0.4 mM NAD+; 2 U each of phosphoglucomutase (Sigma) and Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (Sigma), and up to 100 μl of extract. After a one minute pre-incubation period reactions were initiated by addition of sodium pyrophosphate to 1.5 mM. NADH production was monitored spectrophotometrically at 340 nm against a reaction mixture which lacked sodium pyrophosphate. The rate was proportional to the amount of extract added and the reaction was linear over at least 3 minute time periods. One unit is defined as the amount of enzyme that produces 1 μ mole of glucose-1-P per minute.

Protein concentrations were determined using the dye reagent from BioRad. The results are shown below in Table 2.

TABLE 2 ADP-glucose pyrophosphorylase Activity in Embryos from Lazor and bsg-introgressed lines of Pisum sativum ADPG-PPase activity, Embryo wt, g U/mg protein Lazor .28 .0559 .31 .0373 bsg .28 .0388 .31 .0271

Table 2 shows that the activity of ADP glucose pyrophosphorylase is not greatly reduced in the cotyledons of the present invention when compared to the activity in the variety Lazor.

Analysis of Phosphoglucomutase Isozymes by Starch Gel Electrophoresis

Embryos (0.25-0.3 g) or leaves (0.5 g) frozen in liquid nitrogen were ground in an ice-cold mortar in the presence of extraction buffer (0.1 M Tris-Cl, pH 7.8; 2% reduced glutatione) plus 50 mg of polyvinylpolypyrrolidone and a small amount of acid-washed sand. Extracts were squeezed through one layer of miracloth and, in some experiments, diluted with extraction buffer to equal chlorophyll concentrations (as determined by the formula: [(A₆₄₅×202)+(A₆₆₃×80.2)]×10.5=μg chlorophyll/ml). Extracts were taken up by 5 mm×1 cm 3 MM wicks and placed 4 cm from the cathodal end in a 12% starch gel prepared in 15 mM Tris/4 mM citric acid, pH 7.8. A 3 MM wick was used to connect the starch gel to the electrode buffer (0.3 M sodium borate, pH 7.8). Electrophoresis was done in the cold room for 20 minutes at 200 V/30 mA. The wicks were then removed and electrophoresis was continued at 30 mA until the voltage reached 300 V; the voltage was then maintained at 300 V until the dye front had moved ˜11 cm.

The upper portion of the starch gel was then sliced off and the bottom 1 mm section was stained for phosphoglucomutase (PGM) activity using a 0.7% agarose overlay containing 0.1 M Tris-Cl (pH 7.5), 10 mM MgCl₂, 1.5 mg/ml glucose-1-P, 0.15 mg/ml NADP+, 0.2 mg/ml MTT, 40 μg/ml PMS, and 0.4 U/ml glucose-6 phosphate dehydrogenase. After a 15 minute incubation in the dark at 30° C., the gel was washed for 1 minute in 1% acetic acid, rinsed several times with water, and fixed in 5:2:1:4 ethanol:acetic:acid:glycerine water.

Chloroplasts were isolated according to the method described in Methods in Plant Molecular Biology, 141-172, Cold Spring Harbor Press, Lamppa, J. K., et al., eds. 1995, “Section 8 In vitro Import of Proteins into Chloroplasts,” 1995, herein incorporated by reference. Leaves (˜20 g) were gently homogenized in 450 ml of ice-cold grinding buffer (2 mM EDTA, 1 mM MgCl₂, 1 mM MnCl₂, 50 mM HEPES-KOH (pH 7.5), 0.33 M sorbitol) with a Polytron set at 3.5 for 2 one minute bursts. The homogenate was filtered through four layers of cheesecloth and four layers of Miracloth and then pelleted in a GSA rotor at 4000 rpm for 1 minute at 4° C. The pellets were resuspended in 8 ml of grinding buffer and layered over two 40%/80% Percoll step gradients made up in grinding buffer plus 0.86 mg/ml ascorbic acid and 0.35 mg/ml reduced glutathione. The gradients were spun in an HB-4 rotor at 7500 rpm for 8 minutes at 4° C. The lower chloroplast band was resuspended in 30 ml grinding buffer and repelleted by centrifugation in an HB-4 rotor at 3500 rpm for 5 minutes. The pellets were resuspended in 30 ml of 1×HSM (10 mM HEPES-KOH, pH 8, 0.33 M sorbitol, 8.4 mM methionine) and repelleted in an HB-4 rotor at 3500 rpm for 5 minutes at 4° C. The pellets were then either lysed by being taken up in hypotonic medium (10 mM Tris-Cl, pH 7.8, 2% reduced glutathione) or resuspended in 1×HSM and lysed by freeze-thawing.

FIG. 4 shows the electrophorectic separation of phosphoglucomutase activities in Lazor and in the Pisum sativum line having the bsg gene introgressed into Lazor. In Lazor, two isozymes are resolved in extracts from its leaves, whereas only one isozyme stains for phosphoglucomutase isozymes in the bsg gene introgressed Pisum sativum line of Lazor. In extracts from leaves from the bsg introgressed line of Lazor, the isozyme missing from the bsg-introgressed Pisum sativum line of Lazor, which is the less electronegative isozyme, is the isozyme found in isolated chloroplasts. The same isozyme band is also missing in embryo extracts from the bsg-introgressed Pisum sativum line of Lazor which is shown in FIG. 5.

EXAMPLE 3 Sugar Analysis of Frozen Peas

Peas of a bsg introgressed line, derived from crosses with Encore and referred to as Encore BSG, and peas of the Pisum sativum variety XPF330 were frozen for high performance liquid chromatography (HPLC). XPF330 is a proprietary variety of Seminis Vegetable Seeds, Inc. XPF330 is late season, produces wrinkled mature seed that contains the r gene and is resistant to Powdery Mildew Fungus. The frozen pea samples were stored at −20 degrees centigrade until extracted. The peas were subsampled and weighed out on a Mettler AT200 analytical balance. Absolute ethanol was added to the subsample (4:1 ratio, volume/weight). The sample was cooled and stored overnight (at −20° C.) then ground with an IKA Ultra Turrax T25 homogenizer. The homogenate was returned to the freezer. The following day, the sample was mixed, then centrifuged to clarify the extract. An aliquot was removed and sealed in an autosampler vial and stored at −20° C. until analyzed.

The sugars are separated on a Hewlett-Packard 1050 HPLC chemstation system, employing a refractive index detector. The column used was a Whatman Partsil 5PAC column (4×12.5 mm). The solvent system was composed of 85% acetonitrile at a flow rate of 0.85 ml/minute at 35° C. The injection volume was 5 microliters. A standard mixture of glucose, fructose and sucrose in 80% ethanol was used to quantitate the sugars. This standard was injected after every tenth sample. The results are shown below in Table 3.

TABLE 3 LINE Fru Glc Suc Encore BSG Avg. 0.058 0.031 6.773 Stds 0.008 0.011 0.271 CV 14.451 33.973 4.001 StdErr 0.003 0.004 0.111 Count 6 XPF 330 Avg. 0.037 0.019 4.552 Stds 0.003 0.004 0.125 CV 7.884 22.959 2.757 StdErr 0.001 0.002 0.051 Count 6

The results shown above in Table 3 demonstrate that peas containing the bsg gene homozygous within their genome contain higher levels of fructose, glucose and sucrose than peas containing the r gene homozygous within its genes.

EXAMPLE 4 Sucrose Analysis of Frozen Peas

Frozen samples from the Pisum sativum varieties or lines listed below in Tables 4 and 5 were stored at −20° C. until extracted. The peas were subsampled and weighed out on a Mettler AT200 analytical balance. Absolute ethanol was added to the subsample (4:1 ratio, volume/weight). The sample was cooled and stored overnight (at −20° C.) then ground with an IKA Ultra Turrax T25 homogenizer. The homogenate was returned to the freezer. The following day, the sample was mixed, then centrifuged to clarify the extract. An aliquot was removed and scaled in an autosampler vial and sealed in an autosampler vial and stored at −20° C. until analyzed.

The sugars are separated on a Hewlett-Packard 1050 HPLC chemstation system, employing a refractive index detector. The column used was a Whatman Partsil 5PAC column (4×12.5 mm). The solvent system was composed of 85% acetonitrile at a flow rate of 0.85 ml/minute at 35° C. The injection volume was 5 microliters. A standard mixture of sucrose in 80% ethanol was used to quantitate sucrose. This standard was injected every tenth sample. The results are shown below in Tables 4 and 5.

TABLE 4 (fresh weight) Variety Tenderometer % Sucrose Average bsg gene Bolero 99 4.75 No Bolero 99 4.64 No Dual 97 4.56 No Dual 97 4.27 No EX 8500567 95 6.26 Yes EX 8500567 95 6.22 Yes EX 8500567 102 6.39 Yes EX 8500567 102 6.40 Yes Lazor 93 4.78 No Lazor 93 5.06 No Lazor 99 4.82 No Lazor 99 4.89 No Lazor BSG 95 6.37 Yes Lazor BSG 95 6.50 Yes Lazor BSG 97 6.45 Yes Lazor BSG 97 6.56 Yes Quantum 98 4.82 No Quantum 98 4.83 No Spring 98 4.86 No Spring 104 4.77 No Tacoma 94 4.78 No Tacoma 94 4.72 No Tacoma 99 4.47 No Tacoma 99 4.39 No XP F357 96 5.15 No XP F357 96 5.57 No XP F357 104 5.36 No XP F357 104 5.31 No

TABLE 5 Sucrose Analysis of Frozen Peas (fresh weight) Variety % Sucrose Average Tenderometer bsg gene B1R X MCRO 4.90 97 No BEMAf x Alf 4.81 100 No Bolero 5.25 96 No Bolero x Bemol Af 4.75 98 No CM279 5.52 102 No CM279 5.45 98 No CM279 5.57 98 No CM279 5.02 100 No Dinos 4.74 97 No 8500567 6.59 96 Yes 8500567 7.06 97 Yes 8500567 6.70 90 Yes 8500567 5.75 87 Yes EF680 X Tacoma 4.83 104 No Encore X Tacoma 4.79 98 No F226 X Darfon 4.61 103 No F234 X Mrkd 5.73 103 No F240 X Tacoma 5.16 97 No F240 X Tacoma 4.90 98 No F353 5.50 99 No F357 5.11 97 No F383 5.38 97 No Kalamo 5.09 97 No Lazor 5.61 100 No Lazor 5.31 97 No Lazor 5.25 97 No Lazor BSG 7.10 92 Yes Lazor BSG 7.07 95 Yes Lazor BSG 6.74 93 Yes Lea X F240 5.86 97 No Lea X Tacoma 5.18 103 No M153 4.84 97 No Micro PMB C1 4.88 97 No Novll X F240 5.06 101 No P1Sdin X Nvll 5.42 100 No P1sDn X Nvll 6.08 97 No Sknd X R86275 X 5.13 102 No Sknd X R86275 X 5.51 98 No Snake 5.09 98 No Spr DMBC X 5.85 98 No Spring 5.34 95 No Spring 5.20 99 No Spring 4.78 96 No

The Pisum sativum varieties, Bolero, Dual, Lazor, Quantum, Spring and Tacoma listed in Table 4, produce wrinkled mature seeds and contain r gene homozygous within their genome. These varieties are commercially available from Seminis Vegetable Seeds, Inc. The Pisum sativum varieties 8500567 and 8500557, also shown in Table 4, produce highly wrinkled seeds and contain the bsg gene homozygous within their genome. 8500567 and 8500557 are proprietary varieties of Seminis Vegetable Seeds, Inc. The Pisum sativum line, Lazor BSG also shown in Table 4, produces highly wrinkled seeds and contains the bsg gene homozygous within its genome. Lazor BSG is a proprietary variety of Seminis Vegetable Seeds, Inc. XPF357, also shown in Table 4, produces wrinkled seeds and contains the r gene within its genome. XPF357 is a proprietary variety of Seminis Vegetable Seeds, Inc.

The Pisum sativum varieties and breeding lines shown in Table 5, with the exception of 8000567 and Lazor BSG, produce wrinkled seeds and contain the r gene homozygous within their genome. All of these varieties (with the exception of Snake) are proprietary varieties (or breeding lines) of Seminis Vegetable Seeds, Inc. The Pisum sativum varieties Bolero, Lazor, Dinos, Kalamo and Spring, produce wrinkled seeds and contain the r gene homozygous within their genome. These varieties are commercially available from Seminis Vegetable Seeds, Inc.

Snake, also shown in Table 6, produces wrinkled seeds and contains the r gene homozygous within its genome. Snake is commercially available from Cristes Moscow Growers, Moscow, Id.

The average sucrose produced by peas containing the bsg gene homozygous in its genome in Tables 4 and 5 was 6.54 (with values ranging from 5.75 to 7.10 when measured at tenderometer values from about 87 to about 102). The average amount of sucrose produced by peas that did not contain the bsg gene in the sucrose tests conducted in 1996-1997 was 5.08 (with values ranging from 4.27 to 6.08).

EXAMPLE 5 Alcohol Insoluble Solids Analysis in Various Peas

This Example describes the alcohol insoluble solids content on canned peas from several different Pisum sativum varieties or lines. The protocol for determining the alcohol insoluble solids on these canned peas is as follows:

a) open can, pour the contents over an 8 mesh sieve, rinse with two volumes of tap water, and allowed to drain for two (2) minutes;

b) weigh 90-100 grams of peas to the nearest 0.1 gram, transfer to a blender cup, add to the cup an equal volume of deionized water;

c) blend the peas for two (2) minutes on the blend setting;

d) weigh 40 grams of the blended mix to the nearest 0.01 grams and transfer quantitatively to a 1 liter boiling flask fitted with a reflux condenser. 280 ml of 86% ethanol is used to complete the transfer;

e) boil the mixture for 30 minutes;

f) place a 11 cm Whatman #1 filter paper, previously dried in a metal weighing dish and weighed to the nearest 0.01 grams, into an 8 cm Buchner filter apparatus, in a manner so that the edges of the paper extend 1 cm up the sides of the filter apparatus. Apply suction to the apparatus and filter the alcohol-solids mixture quantitatively through the paper; wash the residue from the flask with 80% ethanol until the washings are colorless. Do not overfill the cup formed by the filter paper; and

g) return the filter paper to a drying oven in its original metal weighing dish and dry at 100° C. for 2 hours. Cool in a desiccator and weigh to the nearest 0.01 grams. Determine the weight of dry, alcohol insoluble solids by difference and multiply by 5 to convert to percentage alcohol insoluble solids.

The alcohol insoluble solids value at 105 tenderometer was calculated using linear regression from paired measures of alcohol insoluble solids and tenderometer for each variety. The tenderometer value at 12% alcohol insoluble solids was calculated by linear regression from the same paired data. For the pea varieties of the present invention that contain the bsg gene, it was necessary to extend the regression line beyond the range of data in order to intercept 12 percent. For example, 8500557, a pea variety containing the bsg gene, had 7.2% AIS at a tenderometer of 105, and the calculated tenderometer for 12% AIS is 199. Table 6, below shows the alcohol insoluble solids value at 105 and tenderometer value 12% AIS for several varieties of Pisum sativum.

TABLE 6 Code Tdr= 105 AIS= 12.0 bsg gene 8500557 AIS= 7.2 Tdr= 199 Yes 8500567 AIS= 7.7 Tdr= 200 Yes Tender AIS= 8.9 Tdr= 124 No Revolution AIS= 9.3 Tdr= 135 No 10 002 AIS= 9.8 Tdr= 127 No 20 028 AIS= 10.1 Tdr= 128 No Zamira AIS= 10.2 Tdr= 123 No Pacha AIS= 10.3 Tdr= 126 No Cabro AIS= 10.3 Tdr= 124 No 20 037 AIS= 10.3 Tdr= 124 No CMG290 AIS= 10.4 Tdr= 123 No Magic AIS= 10.4 Tdr= 119 No Avola AIS= 10.4 Tdr= 125 No 20 036 AIS= 10.5 Tdr= 124 No Fresca AIS= 10.5 Tdr= 121 No 10 003 AIS= 10.5 Tdr= 122 No Bolero AIS= 10.5 Tdr= 120 No Samish AIS= 10.6 Tdr= 121 No Quad AIS= 10.6 Tdr= 119 No Titan AIS= 10.6 Tdr= 121 No 20 029 AIS= 10.7 Tdr= 114 No Renard AIS= 11.0 Tdr= 116 No 20 047 AIS= 11.1 Tdr= 115 No 20 019 AIS= 11.3 Tdr= 119 No Methow AIS= 11.5 Tdr= 110 No 20 035 AIS= 11.8 Tdr= 107 No Catalina AIS= 12.0 Tdr= 105 No Fristo AIS= 12.0 Tdr= 105 No Orlando AIS= 13.2 Tdr= 95 No Kong AIS= NS Tdr= NS No Lazor BSGBC1 AIS= 8.0 Tdr= 194 Yes Lazor BSGBC2 AIS= 8.4 Tdr= 180 Yes 20 078 AIS= 8.7 Tdr= 131 No Paso AIS= 9.0 Tdr= 133 No Mathilde AIS= 9.1 Tdr= 131 No 20 071 AIS= 9.3 Tdr= 125 No 20 089 AIS= 9.5 Tdr= 128 No Globo AIS= 9.5 Tdr= 124 No 20 091 AIS= 9.6 Tdr= 121 No Waverex AIS= 9.6 Tdr= 131 No 20 067 AIS= 9.7 Tdr= 129 No 20 100 AIS= 9.8 Tdr= 122 No 20 101 AIS= 9.9 Tdr= 123 No 20 092 AIS= 10.0 Tdr= 127 No 10 021 AIS= 10.0 Tdr= 121 No 10 025 AIS= 10.1 Tdr= 126 No 20 090 AIS= 10.2 Tdr= 125 No Nitro AIS= 10.2 Tdr= 123 No 10 016 AIS= 10.2 Tdr= 123 No 20 075 AIS= 10.3 Tdr= 124 No 10 015 AIS= 10.3 Tdr= 125 No Darfon AIS= 10.3 Tdr= 120 No Kimo AIS= 10.4 Tdr= 123 No 20 093 AIS= 10.4 Tdr= 121 No R555 AIS= 10.4 Tdr= 127 No 20 068 AIS= 10.4 Tdr= 121 No 20 030 AIS= 10.4 Tdr= 122 No Barle AIS= 10.5 Tdr= 119 No 20 069 AIS= 10.5 Tdr= 117 No 10 020 AIS= 10.5 Tdr= 121 No Brule AIS= 10.6 Tdr= 118 No 20 073 AIS= 10.7 Tdr= 119 No Curico AIS= 10.7 Tdr= 115 No Purser AIS= 10.7 Tdr= 115 No 20 094 AIS= 10.7 Tdr= 120 No 20 116 AIS= 10.7 Tdr= 117 No 20 076 AIS= 10.8 Tdr= 119 No Lynx AIS= 10.9 Tdr= 115 No 20 102 AIS= 10.9 Tdr= 118 No 20 103 AIS= 10.9 Tdr= 118 No Sigra AIS= 10.9 Tdr= 116 No Encore AIS= 11.0 Tdr= 115 No Alamado AIS= 12.0 Tdr= 115 No Markado AIS= 11.0 Tdr= 116 No 20 070 AIS= 11.1 Tdr= 115 No Bolero AIS= 11.1 Tdr= 114 No Spring AIS= 11.1 Tdr= 114 No Tyne AIS= 11.1 Tdr= 116 No Deltafon AIS= 11.2 Tdr= 111 No 10 017 AIS= 11.2 Tdr= 113 No Wolf AIS= 11.2 Tdr= 113 No 20 119 AIS= 11.3 Tdr= 111 No Lazor AIS= 11.4 Tdr= 110 No 20 063 AIS= 11.4 Tdr= 110 No 20 074 AIS= 11.4 Tdr= 113 No 20 087 AIS= 11.4 Tdr= 113 No Camina AIS= 11.4 Tdr= 112 No 20 120 AIS= 11.4 Tdr= 112 No Masterfon AIS= 11.5 Tdr= 111 No 20 118 AIS= 11.5 Tdr= 110 No Vevas AIS= 11.5 Tdr= 110 No 20 106 AIS= 11.6 Tdr= 107 No Snake AIS= 11.6 Tdr= 109 No 20 088 AIS= 11.7 Tdr= 108 No 20 117 AIS= 11.8 Tdr= 107 No 20 072 AIS= 11.9 Tdr= 106 No Prism AIS= 12.0 Tdr= 105 No Dual AIS= 13.0 Tdr= 95 No

The Pisum sativum varieties Tender, Revolution, Avola, Orlando, Markado, Barle, Sigra, Encore, Methow, Alamado, Tyne, Deltafon, Lazor, Camina, Wolf, Masterfon, Veras, Pacha, Bolero, Cabro, Fristo, Paso, Mathilde, Globe, Nitro, Darfon, Kimo, Dual, Prism, Magic, Titan, Renard, Catalina, Kong, Curico and Lynx listed in Table 6, produce wrinkled mature seeds and contain the r gene homozygous within their genome. These varieties are commercially available from Seminis Vegetable Seeds, Inc. The Pisum sativum varieties CMG290, Samish, Quad, Snake and Brule produce wrinkled seed and contain the r gene homozygous within their genome. These Pisum sativum varieties are commercially available from Crites Moscow Growers, Moscow, Id.

The Pisum sativum variety Zamira produces wrinkled mature seeds and contains the r gene homozygous within its genome. Zamira is commercially available from Nunhems Zaden (Haelen, Netherlands).

The Pisum sativum variety Fresca produces wrinkled mature seeds and contains the r gene homozygous within its genome. Fresca is commercially available from Vilmoran (Empire, Calif.).

The Pisum sativum variety Waverex produces wrinkled mature seeds and contains the r gene homozygous within its genome. Waverex is commercially available from Van Waveren (Gottingen, West Germany).

The Pisum sativum variety Purser produces wrinkled mature seeds and contains the r gene homozygous within its genome. Purser is commercially available from Novartis (Boise, Id.).

The Pisum sativum varieties 8500557 and 8500567 produce highly wrinkled mature seeds and contain the bsg gene homozygous within their genome. 8500557 and 8500567 are proprietary varieties of Seminis Vegetable Seeds.

All other Pisum sativum varieties or lines used in Table 6 produce wrinkled mature seeds and contains the r gene homozygous within their genome. All are proprietary varieties of Seminis Vegetable Seeds, Inc.

EXAMPLE 6 Analysis of Sucrose and AIS Levels

22 frozen samples of peas were tested for the percentage (%) sucrose and alcohol insoluble solids. Peas were tested from the Pisum sativum varieties or lines Quantum, Lazor and LazorBSGBC2. Lazor BSGBC2 was selected after 2 backcrosses of the bsg from M82.1 into Lazor (as a recurrent parent). The same procedures used in Examples 3 and 4 to determine and AIS were used in this Example. The results are shown in Table 7.

TABLE 7 Variety Tenderometer % Sucrose % AIS Quantum 92 6.01 8.8 Quantum 100 6.26 9.6 Lazor 95 5.88 9.5 Lazor 101 NA 10.7 Lazor BSGBC2 91 7.01 7.3 Lazor BSGBC2 99 7.07 NA

These results show that peas of the Pisum sativum line Lazor BSGBC2 contained a higher percentage fresh weight of sucrose and lower % of AIS than the Quantum and Lazor varieties.

EXAMPLE 7 Molecular Characterization of the bsg Gene

A. Materials and Methods

1. Preparation of Poly(A)+RNA

RNA was prepared from leaves and cotyledons as described in Dunsmuir et al. (1987) In: Gelvin, S. and Schilperoot, R., eds. Plant Molecular Biology Manual, Vol 9. Plenum, N.Y., pp. 45-59, herein incorporated by reference. Poly(A)+RNA was isolated using a Poly A Tract Kit (Promega).

2. PGM Transcript Analysis

Total and poly (A)+cotyledon RNA were separated electrophoretically on a 1.1% agarose gel containing 6% formaldehyde and prepared in a buffer consisting of 20 mM MOPS (3-[N-morpholino]propane-sulfonic acid), 5 mM sodium acetate, 1 mM EDTA, pH 8. This buffer was also used as the reservoir buffer except the pH was adjusted to 7. RNA was denatured at 55° C. in 65% formamide, 8% formaldehyde, 26 mM MOPS, 6.5 mM sodium acetate, 1.3 mM EDTA, pH 8 before being loaded on to the formaldehyde gel. Following electrophoresis at 4 V/cm, the RNA was transferred to a Duralon-UV membrane (Stratagene) and UV-cross-linked with a Stratagene Stratalinker.

For detection of plastid PGM transcripts, Northern blots were hybridized with a 1.1 kb HindIII fragment from the 3′ half of the wild-type cDNA clone, which was labeled with Klenow using random primers (Feinberg, A. P. et al., Anal. Biochem. 132:6-13). Hybridizations were performed in 0.25 M phosphate buffer (pH 7.4), 7% SDS, 1 mM EDTA, 1% bovine serum albumin at 65° C. in a Robbins Hybridization incubator. Final washes were performed in 0.1×SSC at 65° C.

3. Preparation of First Strand cDNA

First strand cDNA was prepared using Superscript reverse transcriptase II (GibcoBRL). First strand synthesis was primed off of the dT₁₇ adapter primer (GGAGATCTGG TAAGCTTGTT TTTTTTTTTT TTTTT₁₇) (SEQ ID NO:21) according to the instructions of the manufacturer and using 200 ng of poly (A)+RNA. The final product was diluted to 0.5 ml with water.

4. Preparation of Genomic DNA

To prepare genomic DNA 2.5 g of leaves frozen in liquid nitrogen were ground in a mortar in the presence of 4 mls of urea buffer (7 M urea, 0.31 M NaCl, 50 mM Tris-Cl (pH 8), 20 mM EDTA, 1% N-lauroyl sarcosine). Following phenol-chloroform extraction, the DNA was precipitated by the addition of 0.3 mls 5 M ammonium acetate and 4.3 mls isopropanol, washed in 70% ethanol, and resuspended in 4 mls of TE (10 mM Tris-Cl (pH 8), 1 mM EDTA). The DNA was then phenol-chloroform extracted in the presence of 0.2 mls 3 M sodium acetate and precipitated by the addition of two volumes of ethanol. After the DNA was thoroughly washed in 70% ethanol, it was resuspended in 0.5 mls TE. The DNA was precipitated one more time by the addition of 125 μl 7.5 M ammonium acetate and 1 volume of isopropanol, washed in 70% ethanol, and resuspended in 0.5 mls TE.

5. PCR Amplification of cDNA and Genomic Sequences

PGM cDNA was amplified by PCR in a 100 μl volume containing 1× cloned Pfu buffer (Stratagene), 0.2 mM of each dNTP (dGTP, DATP, TTP, and dCTP), 50 pmol of each primer, and 5 μl of first strand cDNA. The reaction mixture was warmed to 94° C. before adding 5 U of Pfu DNA polymerase (Stratagene). After a 6 minute denaturation step (94° C.), the temperature was lowered to 64° C. for 45 seconds and then brought to 72° C. for 4-5 minutes. Samples were then subjected to 31 cycles of denaturation (45 sec, 94° C.), annealing (45 sec, 62° C.), and extension (72° C., 4-5 minutes), with the final extension being carried out for an additional 2 minutes. PCR amplifications were performed on a Perkin Elmer Cetus DNA Thermal Cycler.

Genomic sequences were amplified by PCR in a similar manner except that extension times were increased to six minutes. PCR reactions contained ˜500 ng of genomic DNA.

6. Cloning of PCR-amplified Fragments

PCR-amplified fragments were chloroform extracted to remove the mineral oil overlaying and then ethanol precipitated in the presence of 0.3 M sodium acetate and 10 μl of tRNA.

PCR-amplified fragments being cloned into PCRII-TOPO were first incubated for 10 minutes at 72° C. with Taq polymerase in order to add a 3′ dA overhang. They were then cloned into PCRII-TOPO using a TOPO TA cloning kit (Invitrogen).

Polynucleotide kinase was used to add a 5′ phosphate before cloning PCR-amplified fragments into Smal-cut Bluescript or HindIII Smal-cut Bluescript.

7. Sequencing and Sequence Analysis

Genomic and cDNA clones were first phenol-chloroform extracted and ethanol precipitated before being digested with 20 μg/ml ribonuclease A for 10 minutes at 37° C. The ribonuclease A-treated templates were phenol-chloroform extracted and ethanol precipitated one more time before being used as a template in a thermal cycle sequencing reaction performed using a ThermoSequenase fluorescent primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia). Reactions were primed off of fluorescently-labeled M13 forward and M13 reverse primers. Sequencing was performed on a Licor automated fluorescent sequencer. Sequencing data was analyzed using the program Sequencher.

B. Results

1. Isolation of bsg Plastid PGM cDNA Clones

PCR was used to amplify plastid PGM cDNA from the smooth-seed Pisum sativum variety, called “Frimousse” (hereinafter referred to as “wildtype”) and Frimousse introgressed-bsg (hereinafter referred to as the “mutant”) Poly(A)+seed RNA using the primers PGM-Fp (SEQ ID NO:3), PGM-F91 (SEQ ID NO:4), PGM-Fb (SEQ ID NO:5), PGM-F827 (SEQ ID NO:6), PGM-Rg (SEQ ID NO:7), PGM-R1262 (SEQ ID NO:8), PGM-R1816 (SEQ ID NO:9) and PGM-Rf (SEQ ID NO:10) shown in FIG. 9 (SEQ ID NO:11). Most fragments generated by PCR were identical in size in both the mutant and wildtype tissue. However, PCR products amplified from the mutant cDNA encompassing the 555 base pair internal region between primers PGM-R1262 (SEQ ID NO:8) and PGM-R1816 (SEQ ID NO:9) electrophoresed as three bands. One band was approximately 150 base pairs larger than the wildtype band. The second band was approximately 170 base pairs smaller than the wildtype band. The third band was the same size as the wildtype band (see for instance FIG. 10, the Fb/Rf and Fb/R1816 amplifications). PCR products not spanning this 555 base pair internal region did not differ in size between mutant and wildtype. cDNA fragments amplified with primer combinations PGM-Fb/PGM-R1816 (SEQ ID NOS:5 and 9) and PGM-Fp/PGM-Rf (SEQ ID NOS:3 and 10) were then cloned into PCRII-TOPO (Invitrogen) or SmaI-cut Bluescript (Stratagene) for further examination.

PCR was also used to amplify the plastid PGM genomic sequences from the wildtype and mutant. No difference was seen in the size of the amplified bands. Genomic sequences were amplified using the primers PGM-F827 (SEQ ID NO:6) and PGM-R1816 (SEQ ID NO:9). Genomic sequences amplified with these primers were first digested with HindIII and then cloned into HindIII EcoRV-digested Bluescript.

2. Sequence Analysis of PGM cDNA and Genomic Clones

Wildtype Plastid PGM cDNA Sequence

The sequence of the wildtype plastid PGM cDNA is shown in FIG. 11. The region between primers PGM-Fp (SEQ ID NO:3) and PGM-Rf (SEQ ID NO:10) was sequenced in both directions except for a 132 base pair region which was sequenced in only one direction. The region sequenced includes the entire coding region except for the C-terminal 8 amino acid residues. Two independent clones were sequenced throughout this region and no differences were found between these clones.

Wildtype Plastid PGM Genomic Sequence

A partial sequence of the wildtype plastid PGM gene is shown FIG. 7. This gene was sequenced from the HindIII site encoding amino acid residue 244 to primer PGM-R1816 (SEQ ID NO:9), which encodes amino acid residues 573-580. Sequence was conducted in both directions except for a 77 basepair region between nucleotides 1274-1350. More than one independent clone was sequenced except in the region between nucleotides 1016-1350. Within the sequenced region 12 introns were found ranging in size from 79-337 nucleotides.

Mutant PGM Genomic Sequence

The mutant variant of the plastid PGM gene was sequenced in the same region as the wildtype gene except for a 77 base pair gap between nucleotides 1274 and 1350 which was not sequenced at all (FIG. 6). With the exception of the region between nucleotides 959 and 1430, both strands and more than one independent clone were sequenced. Only one difference was found between the wildtype and mutant sequences in the region sequenced, and that was at nucleotide 1594 where A in the 3′ splice site dinucleotide AG/ was replaced by T (asterisked 6 and 7).

Mutant Plastid PGM cDNA1 Sequence

The largest plastid PGM cDNA amplified in the mutant (hereinafter referred to as “cDNA1”) is shown in FIG. 12. cDNA1 was sequenced between primers PGM-Fp (SEQ ID NO:3) and PGM-Rf (SEQ ID NO:10). Between nucleotides 888 and 1788 it was sequenced in both directions and between nucleotides 694 and 1915 more than one independently-derived clone was sequenced. cDNA1 retains not only the intron with the mutated 3′ splice site (asterisked), but the downstream intron as well (both introns are shown in FIG. 12 in italicized typeface). It is not clear why the downstream intron should be retained. It may be that the mutated intron is normally removed before the downstream intron can be processed or perhaps secondary structure resulting from the retention of the mutated intron prevents processing of the downstream intron.

The retention of the two intron sequences in cDNA1 results in a plastid PGM polypeptide that is immediately truncated by a termination codon near the 5′ end of the first retained intron. This results in the C-terminal 173 amino acids being lost from plastid PGM.

In the Northern blot shown in FIG. 8, the only transcript seen to accumulate in bsg cotyledons is larger than the wildtype transcript. Since cDNA1 was the only plastid PGM cDNA amplified that is substantially larger than the wildtype transcript, it is believe to correspond to the major transcript accumulating in the bsg mutant.

Mutant Plastid PGM cDNA2 Sequence

The next largest plastid PGM cDNA amplified in the bsg mutant (hereinafter “cDNA2”) is shown in FIG. 13. cDNA2 was sequenced in both directions from two independent clones in the region between primers PGM-Fb (SEQ ID NO:5) and PGM-R1816 (SEQ ID NO:9). cDNA2 is roughly the same size as the wildtype sequence but is missing the first 11 nucleotides of the exon directly downstream from the mutated intron. This results from the utilization of a cryptic AG 3′ splice site dinucleotide in the downstream exon. As a result, a frameshift mutation is created and 23 amino acids are substituted from the wrong reading frame for the C-terminal 174 amino acids of plastid PGM. In FIG. 13, the two nucleotides flanking the 11 base pair deletion are shown in bold.

Mutant Plastid PGM cDNA3 Sequence

The smallest plastid PGM cDNA amplified in the bsg mutant (hereinafter “cDNA3”) is shown in FIG. 14. Two independent cDNA3 clones were sequenced between primers PGM-Fp (SEQ ID NO:3) and PGM-Rf (SEQ ID NO:10). Both strands were sequenced in the region between nucleotides 716 and 1565. Plastic PGM cDNA3 is smaller than the wildtype transcript due to a deletion of the 142 base pair exon immediately downstream from the mutated intron. This results from the substitution of the 3′ splice site in the downstream intron for the mutated 3′ splice site. As a result, a frameshift mutation is created and 9 amino acids are substituted for the C-terminal 173 amino acids of plastid PGM. In FIG. 14, the two nucleotides flanking this deleted exon are shown in bold.

21 1 2391 DNA Pisum sativum misc_feature (1274)..(1350) N=A or C or G or T/U 1 aagcttcagt gtggaagtaa tagacccagt ttctgattac ctggagttat tggaggtcaa 60 aataatcttt tttcttcatt ataatccagt agaatgattc atgcaagctc gatctctgtg 120 ttgagagttt tatttcattt catttcgtct aagtttattt gttattattt tttgtgcaga 180 cagtgttcga ttttcagcta atcaaaagtc ttatttcacg gccagatttt aggtacaacc 240 ttaacattta ttccaccata atccaaatct tggcacattg tctagaatct gattgtgtta 300 aattttttta ttttaggttt acatttgatg ccatgcatgc agttgccggt gcttatgcaa 360 cacccatttt cgttgataaa cttggtgcta gtccggtata gttcttcccc ttttactctt 420 gtacatagcg ggtacaagtt tatacggtat tgttgatttt gggtgttaag tatgcaatgt 480 aggattcaat ttcaaatgga atacctttgg aagattttgg acatggtcat cctgatccta 540 atctaacgtg agtttagttt tatattttcg acattgtgtt ttcaatcatt agtaaattgt 600 ttttgattct aatgtttatt gaacagatac gcaaaggatc ttgtcaatat tatgtatgct 660 gaaaacggac ctgattttgg tgccgctagt gatggtatgc gagattttag ttatttttga 720 aatttaactt gtttccgttg ataaatcctt gtgcaacaat gttttgtctg aacccaagag 780 caattggatg agatggtaag agatctcttt cagcttaacc tgaggtcctg agtttgaact 840 cagtcctggg cacgcaacag tgctaaattc tcttgagaga gaactttgcc gtccattgcg 900 gtcctcccca gctcgaggga ttagtctctg cagttgcacg cagaggatac ccgattttta 960 ctgtaaaaaa acaatgtttt ttgtctgcat ttgtttactt gataatgttt atgtatttta 1020 actttcgttt aggtgatggt gatagaaata tgattttggg aacaagtttc ttcgtaactc 1080 cttcagactc tgtagccgtt attgcagcca atgcaaaaga agcgattccg tactttaagg 1140 acagtatcaa ggtagaaagt ttgtgcatat catattattc acaagtattc gttgttgtaa 1200 aacagaagtg tcattgttct gtattgtaat tgcagggtct tgcacgatca atgccgacaa 1260 gcggtgctct agannnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn tctagattat ggttctcgaa tgacagacgg 1380 acctcgggtg caggttccca ctggttggaa attctttggt aatcttatgg atgctggaaa 1440 tctgtcgatt tgcggggaag agagttttgg aacaggttct gaccacattc gtgagaaaga 1500 cggaatctgg taactttctt atttttgtat tgagaatagc gggtcgagca tttatcaaac 1560 attatctaag tttctccgac ttattaatat tattgggctg tattagcttg gctttcgatt 1620 attgctcacc gcaacaaaga cacgaaacca ggggagaaat tggtctctgt gtctgatgtt 1680 gtgaaggagc attgggcaac ctatggtaga aatttctttt ctagatacga ttacgaggtt 1740 ggttttgatg ctgcaattga agttttattt gttgtatcac acactttgaa gttttatttt 1800 tcttttgagt tttgacaaat ataaatatag gaatgtgaat ccgaaggcgc aaataagatg 1860 atagagtacc tacgagagct tttgtcgaag agcaagcctg gtgataagta tggtaagtta 1920 ctcacaacca ctttcttatc acagacacgg agacacggac accaaacacg acattgacat 1980 tggcacgtaa tcattagcat acactttccc tgagtatatt taaagtgtga tgagttttct 2040 tgtacaggaa gttacgtcct ccagtttgcc gatgattata catacactga tcctgtaagt 2100 tcttacaact tcacattctc atcatgttga tttttgtttc ttcaacttac ggtaaatcaa 2160 ccatagttca aattctgatt gaataaaaac atgcaggtag atggaagtgt agtatcaaaa 2220 caaggggttc ggtttgtttt caccgatggt tcaagaatta tttaccgttt atcagtaagt 2280 aacgtctgtt taattactta cccgaaaaat ttatgaaatg aaatattaag tgattactta 2340 cggtgttttt gtttacaggg aacgggttct gctggtgcaa ctgttagagt g 2391 2 2391 DNA Pisum sativum 2 aagcttcagt gtggaagtaa tagacccagt ttctgattac ctggagttat tggaggtcaa 60 aataatcttt tttcttcatt ataatccagt agaatgattc atgcaagctc gatctctgtg 120 ttgagagttt tatttcattt catttcgtct aagtttattt gttattattt tttgtgcaga 180 cagtgttcga ttttcagcta atcaaaagtc ttatttcacg gccagatttt aggtacaacc 240 ttaacattta ttccaccata atccaaatct tggcacattg tctagaatct gattgtgtta 300 aattttttta ttttaggttt acatttgatg ccatgcatgc agttgccggt gcttatgcaa 360 cacccatttt cgttgataaa cttggtgcta gtccggtata gttcttcccc ttttactctt 420 gtacatagcg ggtacaagtt tatacggtat tgttgatttt gggtgttaag tatgcaatgt 480 aggattcaat ttcaaatgga atacctttgg aagattttgg acatggtcat cctgatccta 540 atctaacgtg agtttagttt tatattttcg acattgtgtt ttcaatcatt agtaaattgt 600 ttttgattct aatgtttatt gaacagatac gcaaaggatc ttgtcaatat tatgtatgct 660 gaaaacggac ctgattttgg tgccgctagt gatggtatgc gagattttag ttatttttga 720 aatttaactt gtttccgttg ataaatcctt gtgcaacaat gttttgtctg aacccaagag 780 caattggatg agatggtaag agatctcttt cagcttaacc tgaggtcctg agtttgaact 840 cagtcctggg cacgcaacag tgctaaattc tcttgagaga gaactttgcc gtccattgcg 900 gtcctcccca gctcgaggga ttagtctctg cagttgcacg cagaggatac ccgattttta 960 ctgtaaaaaa acaatgtttt ttgtctgcat ttgtttactt gataatgttt atgtatttta 1020 actttcgttt aggtgatggt gatagaaata tgattttggg aacaagtttc ttcgtaactc 1080 cttcagactc tgtagccgtt attgcagcca atgcaaaaga agcgattccg tactttaagg 1140 acagtatcaa ggtagaaagt ttgtgcatat catattattc acaagtattc gttgttgtaa 1200 aacagaagtg tcattgttct gtattgtaat tgcagggtct tgcacgatca atgccgacaa 1260 gcggtgctct agatagagtt gctgaaaagt tgaacctccc tttttttgag gtatagtatg 1320 attttacatt gttgttgcgt ttagaattat tctagattat ggttctcgaa tgacagacgg 1380 acctcgggtg caggttccca ctggttggaa attctttggt aatcttatgg atgctggaaa 1440 tctgtcgatt tgcggggaag agagttttgg aacaggttct gaccacattc gtgagaaaga 1500 cggaatctgg taactttctt atttttgtat tgagaatagc gggtcgagca tttatcaaac 1560 attatctaag tttctccgac ttattaatat tatagggctg tattagcttg gctttcgatt 1620 attgctcacc gcaacaaaga cacgaaacca ggggagaaat tggtctctgt gtctgatgtt 1680 gtgaaggagc attgggcaac ctatggtaga aatttctttt ctagatacga ttacgaggtt 1740 ggttttgatg ctgcaattga agttttattt gttgtatcac acactttgaa gttttatttt 1800 tcttttgagt tttgacaaat ataaatatag gaatgtgaat ccgaaggcgc aaataagatg 1860 atagagtacc tacgagagct tttgtcgaag agcaagcctg gtgataagta tggtaagtta 1920 ctcacaacca ctttcttatc acagacacgg agacacggac accaaacacg acattgacat 1980 tggcacgtaa tcattagcat acactttccc tgagtatatt taaagtgtga tgagttttct 2040 tgtacaggaa gttacgtcct ccagtttgcc gatgattata catacactga tcctgtaagt 2100 tcttacaact tcacattctc atcatgttga tttttgtttc ttcaacttac ggtaaatcaa 2160 ccatagttca aattctgatt gaataaaaac atgcaggtag atggaagtgt agtatcaaaa 2220 caaggggttc ggtttgtttt caccgatggt tcaagaatta tttaccgttt atcagtaagt 2280 aacgtctgtt taattactta cccgaaaaat ttatgaaatg aaatattaag tgattactta 2340 cggtgttttt gtttacaggg aacgggttct gctggtgcaa ctgttagagt g 2391 3 24 DNA Pisum sativum 3 cactgttaca gactcgatca atgg 24 4 24 DNA Pisum sativum 4 cagactcgac aacttcatca tctc 24 5 24 DNA Pisum sativum 5 gattgctgat attcccgatg ttga 24 6 24 DNA Pisum sativum 6 gacccagttt ctgattacct ggag 24 7 24 DNA Pisum sativum 7 catgcatggc atcaaatgta aacc 24 8 24 DNA Pisum sativum 8 gcattgatcg tgcaagaccc ttga 24 9 24 DNA Pisum sativum 9 cactctaaca gttgcaccag caga 24 10 24 DNA Pisum sativum 10 tccctgtgaa gtctttgagc tttg 24 11 2182 DNA Pisum sativum CDS (77)..(1954) 11 caaacacata gttaaacaaa aaacactctc tcttgactct tcgaagaaaa agttgtcact 60 gttacagact cgatca atg gct ttc tgt tac aga ctc gac aac ttc atc atc 112 Met Ala Phe Cys Tyr Arg Leu Asp Asn Phe Ile Ile 1 5 10 tct gcg ttt aaa ccc aaa cac tca aat gtc cca ctt tca att cat cat 160 Ser Ala Phe Lys Pro Lys His Ser Asn Val Pro Leu Ser Ile His His 15 20 25 tca tca tcc aat ttt cct tct ttc aaa gtt caa aac ttt cct ttc agg 208 Ser Ser Ser Asn Phe Pro Ser Phe Lys Val Gln Asn Phe Pro Phe Arg 30 35 40 gtt cgc tat aat tca gct att aga gcc act tca tct tcc tct tct act 256 Val Arg Tyr Asn Ser Ala Ile Arg Ala Thr Ser Ser Ser Ser Ser Thr 45 50 55 60 ccc aca acc att gca gaa cct aat gac att aag att aac tct att cct 304 Pro Thr Thr Ile Ala Glu Pro Asn Asp Ile Lys Ile Asn Ser Ile Pro 65 70 75 act aaa cct att gaa gaa caa aaa act ggt acc agt ggt cta aga aaa 352 Thr Lys Pro Ile Glu Glu Gln Lys Thr Gly Thr Ser Gly Leu Arg Lys 80 85 90 aag gtg aaa gtg ttt aag caa gaa aat tac ctt gca aat tgg att cag 400 Lys Val Lys Val Phe Lys Gln Glu Asn Tyr Leu Ala Asn Trp Ile Gln 95 100 105 gca ctg ttt aat tcg ttg ccg ccg gag gat tac aag aat gga ttg ttg 448 Ala Leu Phe Asn Ser Leu Pro Pro Glu Asp Tyr Lys Asn Gly Leu Leu 110 115 120 gtt ttg gga ggc gat ggt cga tac ttc aat aaa gaa gct gca cag ata 496 Val Leu Gly Gly Asp Gly Arg Tyr Phe Asn Lys Glu Ala Ala Gln Ile 125 130 135 140 ata atc aag att gct gct gga aat ggt gtt gga aaa att ctg gtt ggg 544 Ile Ile Lys Ile Ala Ala Gly Asn Gly Val Gly Lys Ile Leu Val Gly 145 150 155 aag gaa ggg ata ttg tca acg cca gcc gtt tct gct gtg ata agg aag 592 Lys Glu Gly Ile Leu Ser Thr Pro Ala Val Ser Ala Val Ile Arg Lys 160 165 170 aga gag gca aat ggt ggg ttt atc atg agt gcg agc cat aac cct ggt 640 Arg Glu Ala Asn Gly Gly Phe Ile Met Ser Ala Ser His Asn Pro Gly 175 180 185 gga cct gaa tat gat tgg ggt att aag ttt aat tac agt agc gga caa 688 Gly Pro Glu Tyr Asp Trp Gly Ile Lys Phe Asn Tyr Ser Ser Gly Gln 190 195 200 cct gca cca gaa tcc atc acc gac aag att tac gga aac acc cta tcg 736 Pro Ala Pro Glu Ser Ile Thr Asp Lys Ile Tyr Gly Asn Thr Leu Ser 205 210 215 220 att tct gag ata aag att gct gat att ccc gat gtt gac tta tca aat 784 Ile Ser Glu Ile Lys Ile Ala Asp Ile Pro Asp Val Asp Leu Ser Asn 225 230 235 gtt gga gtt acg aaa ttc gga agc ttc agt gtg gaa gta ata gac cca 832 Val Gly Val Thr Lys Phe Gly Ser Phe Ser Val Glu Val Ile Asp Pro 240 245 250 gtt tct gat tac ctg gag tta ttg gag aca gtg ttc gat ttt cag cta 880 Val Ser Asp Tyr Leu Glu Leu Leu Glu Thr Val Phe Asp Phe Gln Leu 255 260 265 atc aaa agt ctt att tca cgg cca gat ttt agg ttt aca ttt gat gcc 928 Ile Lys Ser Leu Ile Ser Arg Pro Asp Phe Arg Phe Thr Phe Asp Ala 270 275 280 atg cat gca gtt gcc ggt gct tat gca aca ccc att ttc gtt gat aaa 976 Met His Ala Val Ala Gly Ala Tyr Ala Thr Pro Ile Phe Val Asp Lys 285 290 295 300 ctt ggt gct agt ccg gat tca att tca aat gga ata cct ttg gaa gat 1024 Leu Gly Ala Ser Pro Asp Ser Ile Ser Asn Gly Ile Pro Leu Glu Asp 305 310 315 ttt gga cat ggt cat cct gat cct aat cta aca tac gca aag gat ctt 1072 Phe Gly His Gly His Pro Asp Pro Asn Leu Thr Tyr Ala Lys Asp Leu 320 325 330 gtc aat att atg tat gct gaa aac gga cct gat ttt ggt gcc gct agt 1120 Val Asn Ile Met Tyr Ala Glu Asn Gly Pro Asp Phe Gly Ala Ala Ser 335 340 345 gat ggt gat ggt gat aga aat atg att ttg gga aca agt ttc ttc gta 1168 Asp Gly Asp Gly Asp Arg Asn Met Ile Leu Gly Thr Ser Phe Phe Val 350 355 360 act cct tca gac tct gta gcc gtt att gca gcc aat gca aaa gaa gcg 1216 Thr Pro Ser Asp Ser Val Ala Val Ile Ala Ala Asn Ala Lys Glu Ala 365 370 375 380 att ccg tac ttt aag gac agt atc aag ggt ctt gca cga tca atg ccg 1264 Ile Pro Tyr Phe Lys Asp Ser Ile Lys Gly Leu Ala Arg Ser Met Pro 385 390 395 aca agc ggt gct cta gat aga gtt gct gaa aag ttg aac ctc cct ttt 1312 Thr Ser Gly Ala Leu Asp Arg Val Ala Glu Lys Leu Asn Leu Pro Phe 400 405 410 ttt gag gtt ccc act ggt tgg aaa ttc ttt ggt aat ctt atg gat gct 1360 Phe Glu Val Pro Thr Gly Trp Lys Phe Phe Gly Asn Leu Met Asp Ala 415 420 425 gga aat ctg tcg att tgc ggg gaa gag agt ttt gga aca ggt tct gac 1408 Gly Asn Leu Ser Ile Cys Gly Glu Glu Ser Phe Gly Thr Gly Ser Asp 430 435 440 cac att cgt gag aaa gac gga atc tgg gct gta tta gct tgg ctt tcg 1456 His Ile Arg Glu Lys Asp Gly Ile Trp Ala Val Leu Ala Trp Leu Ser 445 450 455 460 att att gct cac cgc aac aaa gac acg aaa cca ggg gag aaa ttg gtc 1504 Ile Ile Ala His Arg Asn Lys Asp Thr Lys Pro Gly Glu Lys Leu Val 465 470 475 tct gtg tct gat gtt gtg aag gag cat tgg gca acc tat ggt aga aat 1552 Ser Val Ser Asp Val Val Lys Glu His Trp Ala Thr Tyr Gly Arg Asn 480 485 490 ttc ttt tct aga tac gat tac gag gaa tgt gaa tcc gaa ggc gca aat 1600 Phe Phe Ser Arg Tyr Asp Tyr Glu Glu Cys Glu Ser Glu Gly Ala Asn 495 500 505 aag atg ata gag tac cta cga gag ctt ttg tcg aag agc aag cct ggt 1648 Lys Met Ile Glu Tyr Leu Arg Glu Leu Leu Ser Lys Ser Lys Pro Gly 510 515 520 gat aag tat gga agt tac gtc ctc cag ttt gcc gat gat tat aca tac 1696 Asp Lys Tyr Gly Ser Tyr Val Leu Gln Phe Ala Asp Asp Tyr Thr Tyr 525 530 535 540 act gat cct gta gat gga agt gta gta tca aaa caa ggg gtt cgg ttt 1744 Thr Asp Pro Val Asp Gly Ser Val Val Ser Lys Gln Gly Val Arg Phe 545 550 555 gtt ttc acc gat ggt tca aga att att tac cgt tta tca gga acg ggt 1792 Val Phe Thr Asp Gly Ser Arg Ile Ile Tyr Arg Leu Ser Gly Thr Gly 560 565 570 tct gct ggt gca act gtt aga gtg tat atc gaa cag ttt gaa cca gat 1840 Ser Ala Gly Ala Thr Val Arg Val Tyr Ile Glu Gln Phe Glu Pro Asp 575 580 585 gtt tct aaa cac gac gtc gat gct caa att gcc ttg aaa cca tta ata 1888 Val Ser Lys His Asp Val Asp Ala Gln Ile Ala Leu Lys Pro Leu Ile 590 595 600 gat tta gca tta tct gtt tca aag ctc aaa gac ttc aca ggg aga gag 1936 Asp Leu Ala Leu Ser Val Ser Lys Leu Lys Asp Phe Thr Gly Arg Glu 605 610 615 620 aag cct aca gtc atc act taatataagt ttggtttttc attttcagtt 1984 Lys Pro Thr Val Ile Thr 625 ttggttattt ttccactttg gagcttagca tcttttttgt ataatatgat attttgtatt 2044 tactttcaag aaaatgaagt atcattgtgt aacagaataa ataatggtat taataatagc 2104 tagcttctat gcagagaagt tgttcttttc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2164 aaaaaaaaaa aaaaaaaa 2182 12 626 PRT Pisum sativum 12 Met Ala Phe Cys Tyr Arg Leu Asp Asn Phe Ile Ile Ser Ala Phe Lys 1 5 10 15 Pro Lys His Ser Asn Val Pro Leu Ser Ile His His Ser Ser Ser Asn 20 25 30 Phe Pro Ser Phe Lys Val Gln Asn Phe Pro Phe Arg Val Arg Tyr Asn 35 40 45 Ser Ala Ile Arg Ala Thr Ser Ser Ser Ser Ser Thr Pro Thr Thr Ile 50 55 60 Ala Glu Pro Asn Asp Ile Lys Ile Asn Ser Ile Pro Thr Lys Pro Ile 65 70 75 80 Glu Glu Gln Lys Thr Gly Thr Ser Gly Leu Arg Lys Lys Val Lys Val 85 90 95 Phe Lys Gln Glu Asn Tyr Leu Ala Asn Trp Ile Gln Ala Leu Phe Asn 100 105 110 Ser Leu Pro Pro Glu Asp Tyr Lys Asn Gly Leu Leu Val Leu Gly Gly 115 120 125 Asp Gly Arg Tyr Phe Asn Lys Glu Ala Ala Gln Ile Ile Ile Lys Ile 130 135 140 Ala Ala Gly Asn Gly Val Gly Lys Ile Leu Val Gly Lys Glu Gly Ile 145 150 155 160 Leu Ser Thr Pro Ala Val Ser Ala Val Ile Arg Lys Arg Glu Ala Asn 165 170 175 Gly Gly Phe Ile Met Ser Ala Ser His Asn Pro Gly Gly Pro Glu Tyr 180 185 190 Asp Trp Gly Ile Lys Phe Asn Tyr Ser Ser Gly Gln Pro Ala Pro Glu 195 200 205 Ser Ile Thr Asp Lys Ile Tyr Gly Asn Thr Leu Ser Ile Ser Glu Ile 210 215 220 Lys Ile Ala Asp Ile Pro Asp Val Asp Leu Ser Asn Val Gly Val Thr 225 230 235 240 Lys Phe Gly Ser Phe Ser Val Glu Val Ile Asp Pro Val Ser Asp Tyr 245 250 255 Leu Glu Leu Leu Glu Thr Val Phe Asp Phe Gln Leu Ile Lys Ser Leu 260 265 270 Ile Ser Arg Pro Asp Phe Arg Phe Thr Phe Asp Ala Met His Ala Val 275 280 285 Ala Gly Ala Tyr Ala Thr Pro Ile Phe Val Asp Lys Leu Gly Ala Ser 290 295 300 Pro Asp Ser Ile Ser Asn Gly Ile Pro Leu Glu Asp Phe Gly His Gly 305 310 315 320 His Pro Asp Pro Asn Leu Thr Tyr Ala Lys Asp Leu Val Asn Ile Met 325 330 335 Tyr Ala Glu Asn Gly Pro Asp Phe Gly Ala Ala Ser Asp Gly Asp Gly 340 345 350 Asp Arg Asn Met Ile Leu Gly Thr Ser Phe Phe Val Thr Pro Ser Asp 355 360 365 Ser Val Ala Val Ile Ala Ala Asn Ala Lys Glu Ala Ile Pro Tyr Phe 370 375 380 Lys Asp Ser Ile Lys Gly Leu Ala Arg Ser Met Pro Thr Ser Gly Ala 385 390 395 400 Leu Asp Arg Val Ala Glu Lys Leu Asn Leu Pro Phe Phe Glu Val Pro 405 410 415 Thr Gly Trp Lys Phe Phe Gly Asn Leu Met Asp Ala Gly Asn Leu Ser 420 425 430 Ile Cys Gly Glu Glu Ser Phe Gly Thr Gly Ser Asp His Ile Arg Glu 435 440 445 Lys Asp Gly Ile Trp Ala Val Leu Ala Trp Leu Ser Ile Ile Ala His 450 455 460 Arg Asn Lys Asp Thr Lys Pro Gly Glu Lys Leu Val Ser Val Ser Asp 465 470 475 480 Val Val Lys Glu His Trp Ala Thr Tyr Gly Arg Asn Phe Phe Ser Arg 485 490 495 Tyr Asp Tyr Glu Glu Cys Glu Ser Glu Gly Ala Asn Lys Met Ile Glu 500 505 510 Tyr Leu Arg Glu Leu Leu Ser Lys Ser Lys Pro Gly Asp Lys Tyr Gly 515 520 525 Ser Tyr Val Leu Gln Phe Ala Asp Asp Tyr Thr Tyr Thr Asp Pro Val 530 535 540 Asp Gly Ser Val Val Ser Lys Gln Gly Val Arg Phe Val Phe Thr Asp 545 550 555 560 Gly Ser Arg Ile Ile Tyr Arg Leu Ser Gly Thr Gly Ser Ala Gly Ala 565 570 575 Thr Val Arg Val Tyr Ile Glu Gln Phe Glu Pro Asp Val Ser Lys His 580 585 590 Asp Val Asp Ala Gln Ile Ala Leu Lys Pro Leu Ile Asp Leu Ala Leu 595 600 605 Ser Val Ser Lys Leu Lys Asp Phe Thr Gly Arg Glu Lys Pro Thr Val 610 615 620 Ile Thr 625 13 1875 DNA Pisum sativum CDS (21)..(1874) 13 cactgttaca gactcgatca atg gct ttc tgt tac aga ctc gac aac ttc atc 53 Met Ala Phe Cys Tyr Arg Leu Asp Asn Phe Ile 1 5 10 atc tct gcg ttt aaa ccc aaa cac tca aat gtc cca ctt tca att cat 101 Ile Ser Ala Phe Lys Pro Lys His Ser Asn Val Pro Leu Ser Ile His 15 20 25 cat tca tca tcc aat ttt cct tct ttc aaa gtt caa aac ttt cct ttc 149 His Ser Ser Ser Asn Phe Pro Ser Phe Lys Val Gln Asn Phe Pro Phe 30 35 40 agg gtt cgc tat aat tca gct att aga gcc act tcg tct tcc tct tct 197 Arg Val Arg Tyr Asn Ser Ala Ile Arg Ala Thr Ser Ser Ser Ser Ser 45 50 55 act ccc aca acc att gca gaa cct aat gac att aag att aac tct att 245 Thr Pro Thr Thr Ile Ala Glu Pro Asn Asp Ile Lys Ile Asn Ser Ile 60 65 70 75 cct act aaa cct att gaa gga caa aaa act ggt acc agt ggc cta aga 293 Pro Thr Lys Pro Ile Glu Gly Gln Lys Thr Gly Thr Ser Gly Leu Arg 80 85 90 aaa aag gtg aaa gtg ttt aag caa gaa aat tac ctt gca aat tgg att 341 Lys Lys Val Lys Val Phe Lys Gln Glu Asn Tyr Leu Ala Asn Trp Ile 95 100 105 cag gca ctg ttt aat tcg ttg ccg ccg gag gat tac aag aat gga ttg 389 Gln Ala Leu Phe Asn Ser Leu Pro Pro Glu Asp Tyr Lys Asn Gly Leu 110 115 120 ttg gtt ttg gga ggc gat ggt cga tac ttc aat aaa gaa gct gca cag 437 Leu Val Leu Gly Gly Asp Gly Arg Tyr Phe Asn Lys Glu Ala Ala Gln 125 130 135 ata ata atc aag att gct gct gga aat ggt gtt gga aaa att ctg gtt 485 Ile Ile Ile Lys Ile Ala Ala Gly Asn Gly Val Gly Lys Ile Leu Val 140 145 150 155 ggg aag gaa ggg ata ttg tca acg cca gcc gtt tct gct gtg ata agg 533 Gly Lys Glu Gly Ile Leu Ser Thr Pro Ala Val Ser Ala Val Ile Arg 160 165 170 aag aga gag gca aat ggt ggg ttt atc atg agt gcg agc cat aac cct 581 Lys Arg Glu Ala Asn Gly Gly Phe Ile Met Ser Ala Ser His Asn Pro 175 180 185 ggt gga cct gaa tat gat tgg ggt att aag ttt aat tac agt agc gga 629 Gly Gly Pro Glu Tyr Asp Trp Gly Ile Lys Phe Asn Tyr Ser Ser Gly 190 195 200 caa cct gca cca gaa tcc atc acc gac aag att tac gga aac acc cta 677 Gln Pro Ala Pro Glu Ser Ile Thr Asp Lys Ile Tyr Gly Asn Thr Leu 205 210 215 tcg att tct gag ata aag att gct gat att ccc gat gtt gac tta tca 725 Ser Ile Ser Glu Ile Lys Ile Ala Asp Ile Pro Asp Val Asp Leu Ser 220 225 230 235 aat gtt gga gtt acg aaa ttc gga agc ttc agt gtg gaa gta ata gac 773 Asn Val Gly Val Thr Lys Phe Gly Ser Phe Ser Val Glu Val Ile Asp 240 245 250 cca gtt tct gat tac ctg gag tta ttg gag aca gtg ttc gat ttt cag 821 Pro Val Ser Asp Tyr Leu Glu Leu Leu Glu Thr Val Phe Asp Phe Gln 255 260 265 cta atc aaa agt ctt att tca cgg cca gat ttt agg ttt aca ttt gat 869 Leu Ile Lys Ser Leu Ile Ser Arg Pro Asp Phe Arg Phe Thr Phe Asp 270 275 280 gcc atg cat gca gtt gcc ggt gct tat gca aca ccc att ttc gtt gat 917 Ala Met His Ala Val Ala Gly Ala Tyr Ala Thr Pro Ile Phe Val Asp 285 290 295 aaa ctt ggt gct agt ccg gat tca att tca aat gga ata cct ttg gaa 965 Lys Leu Gly Ala Ser Pro Asp Ser Ile Ser Asn Gly Ile Pro Leu Glu 300 305 310 315 gat ttt gga cat ggt cat cct gat cct aat cta aca tac gca aag gat 1013 Asp Phe Gly His Gly His Pro Asp Pro Asn Leu Thr Tyr Ala Lys Asp 320 325 330 ctt gtc aat att atg tat gct gaa aac gga cct gat ttt ggt gcc gct 1061 Leu Val Asn Ile Met Tyr Ala Glu Asn Gly Pro Asp Phe Gly Ala Ala 335 340 345 agt gat ggt gat ggt gat aga aat atg att ttg gga aca agt ttc ttc 1109 Ser Asp Gly Asp Gly Asp Arg Asn Met Ile Leu Gly Thr Ser Phe Phe 350 355 360 gta act cct tca gac tct gta gcc gtt att gca gcc aat gca aaa gaa 1157 Val Thr Pro Ser Asp Ser Val Ala Val Ile Ala Ala Asn Ala Lys Glu 365 370 375 gcg att ccg tac ttt aag gac agt atc aag ggt ctt gca cga tca atg 1205 Ala Ile Pro Tyr Phe Lys Asp Ser Ile Lys Gly Leu Ala Arg Ser Met 380 385 390 395 ccg aca agc ggt gct cta gat aga gtt gct gaa aag ttg aac ctc cct 1253 Pro Thr Ser Gly Ala Leu Asp Arg Val Ala Glu Lys Leu Asn Leu Pro 400 405 410 ttt ttt gag gtt ccc act ggt tgg aaa ttc ttt ggt aat ctt atg gat 1301 Phe Phe Glu Val Pro Thr Gly Trp Lys Phe Phe Gly Asn Leu Met Asp 415 420 425 gct gga aat ctg tcg att tgc ggg gaa gag agt ttt gga aca ggt tct 1349 Ala Gly Asn Leu Ser Ile Cys Gly Glu Glu Ser Phe Gly Thr Gly Ser 430 435 440 gac cac att cgt gag aaa gac gga atc tgg gct gta tta gct tgg ctt 1397 Asp His Ile Arg Glu Lys Asp Gly Ile Trp Ala Val Leu Ala Trp Leu 445 450 455 tcg att att gct cac cgc aac aaa gac acg aaa cca ggg gag aaa ttg 1445 Ser Ile Ile Ala His Arg Asn Lys Asp Thr Lys Pro Gly Glu Lys Leu 460 465 470 475 gtc tct gtg tct gat gtt gtg aag gag cat tgg gca acc tat ggt aga 1493 Val Ser Val Ser Asp Val Val Lys Glu His Trp Ala Thr Tyr Gly Arg 480 485 490 aat ttc ttt tct aga tac gat tac gag gaa tgt gaa tcc gaa ggc gca 1541 Asn Phe Phe Ser Arg Tyr Asp Tyr Glu Glu Cys Glu Ser Glu Gly Ala 495 500 505 aat aag atg ata gag tac cta cga gag ctt ttg tcg aag agc aag cct 1589 Asn Lys Met Ile Glu Tyr Leu Arg Glu Leu Leu Ser Lys Ser Lys Pro 510 515 520 ggt gat aag tat gga agt tac gtc ctc cag ttt gcc gat gat tat aca 1637 Gly Asp Lys Tyr Gly Ser Tyr Val Leu Gln Phe Ala Asp Asp Tyr Thr 525 530 535 tac act gat cct gta gat gga agt gta gta tca aaa caa ggg gtt cgg 1685 Tyr Thr Asp Pro Val Asp Gly Ser Val Val Ser Lys Gln Gly Val Arg 540 545 550 555 ttt gtt ttc acc gat ggt tca aga att att tac cgt tta tca gga acg 1733 Phe Val Phe Thr Asp Gly Ser Arg Ile Ile Tyr Arg Leu Ser Gly Thr 560 565 570 ggt tct gct ggt gca act gtt aga gtg tat atc gaa cag ttt gaa cca 1781 Gly Ser Ala Gly Ala Thr Val Arg Val Tyr Ile Glu Gln Phe Glu Pro 575 580 585 gat gtt tct aaa cac gac gtc gat gct caa att gcc ttg aaa cca tta 1829 Asp Val Ser Lys His Asp Val Asp Ala Gln Ile Ala Leu Lys Pro Leu 590 595 600 ata gat tta gca tta tct gtt tca aag ctc aaa gac ttc aca ggg a 1875 Ile Asp Leu Ala Leu Ser Val Ser Lys Leu Lys Asp Phe Thr Gly 605 610 615 14 618 PRT Pisum sativum 14 Met Ala Phe Cys Tyr Arg Leu Asp Asn Phe Ile Ile Ser Ala Phe Lys 1 5 10 15 Pro Lys His Ser Asn Val Pro Leu Ser Ile His His Ser Ser Ser Asn 20 25 30 Phe Pro Ser Phe Lys Val Gln Asn Phe Pro Phe Arg Val Arg Tyr Asn 35 40 45 Ser Ala Ile Arg Ala Thr Ser Ser Ser Ser Ser Thr Pro Thr Thr Ile 50 55 60 Ala Glu Pro Asn Asp Ile Lys Ile Asn Ser Ile Pro Thr Lys Pro Ile 65 70 75 80 Glu Gly Gln Lys Thr Gly Thr Ser Gly Leu Arg Lys Lys Val Lys Val 85 90 95 Phe Lys Gln Glu Asn Tyr Leu Ala Asn Trp Ile Gln Ala Leu Phe Asn 100 105 110 Ser Leu Pro Pro Glu Asp Tyr Lys Asn Gly Leu Leu Val Leu Gly Gly 115 120 125 Asp Gly Arg Tyr Phe Asn Lys Glu Ala Ala Gln Ile Ile Ile Lys Ile 130 135 140 Ala Ala Gly Asn Gly Val Gly Lys Ile Leu Val Gly Lys Glu Gly Ile 145 150 155 160 Leu Ser Thr Pro Ala Val Ser Ala Val Ile Arg Lys Arg Glu Ala Asn 165 170 175 Gly Gly Phe Ile Met Ser Ala Ser His Asn Pro Gly Gly Pro Glu Tyr 180 185 190 Asp Trp Gly Ile Lys Phe Asn Tyr Ser Ser Gly Gln Pro Ala Pro Glu 195 200 205 Ser Ile Thr Asp Lys Ile Tyr Gly Asn Thr Leu Ser Ile Ser Glu Ile 210 215 220 Lys Ile Ala Asp Ile Pro Asp Val Asp Leu Ser Asn Val Gly Val Thr 225 230 235 240 Lys Phe Gly Ser Phe Ser Val Glu Val Ile Asp Pro Val Ser Asp Tyr 245 250 255 Leu Glu Leu Leu Glu Thr Val Phe Asp Phe Gln Leu Ile Lys Ser Leu 260 265 270 Ile Ser Arg Pro Asp Phe Arg Phe Thr Phe Asp Ala Met His Ala Val 275 280 285 Ala Gly Ala Tyr Ala Thr Pro Ile Phe Val Asp Lys Leu Gly Ala Ser 290 295 300 Pro Asp Ser Ile Ser Asn Gly Ile Pro Leu Glu Asp Phe Gly His Gly 305 310 315 320 His Pro Asp Pro Asn Leu Thr Tyr Ala Lys Asp Leu Val Asn Ile Met 325 330 335 Tyr Ala Glu Asn Gly Pro Asp Phe Gly Ala Ala Ser Asp Gly Asp Gly 340 345 350 Asp Arg Asn Met Ile Leu Gly Thr Ser Phe Phe Val Thr Pro Ser Asp 355 360 365 Ser Val Ala Val Ile Ala Ala Asn Ala Lys Glu Ala Ile Pro Tyr Phe 370 375 380 Lys Asp Ser Ile Lys Gly Leu Ala Arg Ser Met Pro Thr Ser Gly Ala 385 390 395 400 Leu Asp Arg Val Ala Glu Lys Leu Asn Leu Pro Phe Phe Glu Val Pro 405 410 415 Thr Gly Trp Lys Phe Phe Gly Asn Leu Met Asp Ala Gly Asn Leu Ser 420 425 430 Ile Cys Gly Glu Glu Ser Phe Gly Thr Gly Ser Asp His Ile Arg Glu 435 440 445 Lys Asp Gly Ile Trp Ala Val Leu Ala Trp Leu Ser Ile Ile Ala His 450 455 460 Arg Asn Lys Asp Thr Lys Pro Gly Glu Lys Leu Val Ser Val Ser Asp 465 470 475 480 Val Val Lys Glu His Trp Ala Thr Tyr Gly Arg Asn Phe Phe Ser Arg 485 490 495 Tyr Asp Tyr Glu Glu Cys Glu Ser Glu Gly Ala Asn Lys Met Ile Glu 500 505 510 Tyr Leu Arg Glu Leu Leu Ser Lys Ser Lys Pro Gly Asp Lys Tyr Gly 515 520 525 Ser Tyr Val Leu Gln Phe Ala Asp Asp Tyr Thr Tyr Thr Asp Pro Val 530 535 540 Asp Gly Ser Val Val Ser Lys Gln Gly Val Arg Phe Val Phe Thr Asp 545 550 555 560 Gly Ser Arg Ile Ile Tyr Arg Leu Ser Gly Thr Gly Ser Ala Gly Ala 565 570 575 Thr Val Arg Val Tyr Ile Glu Gln Phe Glu Pro Asp Val Ser Lys His 580 585 590 Asp Val Asp Ala Gln Ile Ala Leu Lys Pro Leu Ile Asp Leu Ala Leu 595 600 605 Ser Val Ser Lys Leu Lys Asp Phe Thr Gly 610 615 15 2006 DNA Pisum sativum misc_feature (1976)..(1976) N = A or C or G or T/U 15 ctttctgtta cagactcgac aacttcatca tctctgcgtt taaacccaaa cactcaaatg 60 tcccactttc aattcatcat tcatcatcca attttccttc tttcaaagtt caaaactttc 120 ctttcagggt tcgctataat tcagctatta gagccacttc gtcttcctct tctactccca 180 caaccattgc agaacctaat gacattaaga ttaactctat tcctactaaa cctattgaag 240 gacaaaaaac tggtaccagt ggcctaagaa aaaaggtgaa agtgtttaag caagaaaatt 300 accttgcaaa ttggattcag gcactgttta attcgttgcc gccggaggat tacaagaatg 360 gattgttggt tttgggaggc gatggtcgat acttcaataa agaagctgca cagataataa 420 tcaagattgc tgctggaaat ggtgttggaa aaattctggt tgggaaggaa gggatattgt 480 caacgccagc cgtttctgct gtgataagga agagagaggc aaatggtggg tttatcatga 540 gtgcgagcca taaccctggt ggacctgaat atgattgggg tattaagttt aattacagta 600 gcggacaacc tgcaccagaa tccatcaccg acaagattta cggaaacacc ctatcgattt 660 ctgagataaa gattgctgat attcccgatg ttgacttatc aaatgttgga gttacgaaat 720 tcggaagctt cagtgtggaa gtaatagacc cagtttctga ttacctggag ttattggaga 780 cagtgttcga ttttcagcta atcaaaagtc ttatttcacg gccagatttt aggtttacat 840 ttgatgccat gcatgcagtt gccggtgctt atgcaacacc cattttcgtt gataaacttg 900 gtgctagtcc ggattcaatt tcaaatggaa tacctttgga agattttgga catggtcatc 960 ctgatcctaa tctaacatac gcaaaggatc ttgtcaatat tatgtatgct gaaaacggac 1020 ctgattttgg tgccgctagt gatggtgatg gtgatagaaa tatgattttg ggaacaagtt 1080 tcttcgtaac tccttcagac tctgtagccg ttattgcagc caatgcaaaa gaagcgattc 1140 cgtactttaa ggacagtatc aagggtcttg cacgatcaat gccgacaagc ggtgctctag 1200 atagagttgc tgaaaagttg aacctccctt tttttgaggt tcccactggt tggaaattct 1260 ttggtaatct tatggatgct ggaaatctgt cgatttgcgg ggaagagagt tttggaacag 1320 gttctgacca cattcgtgag aaagacggaa tctggtaact ttcttatttt tgtattgaga 1380 atagcgggtc gagcatttat caaacattat ctaagtttct ccgacttatt aatattattg 1440 ggctgtatta gcttggcttt cgattattgc tcaccgcaac aaagacacga aaccagggga 1500 gaaattggtc tctgtgtctg atgttgtgaa ggagcattgg gcaacctatg gtagaaattt 1560 cttttctaga tacgattacg aggttggttt tgatgctgca attgaagttt tatttgttgt 1620 atcacacact ttgaagtttt atttttcttt tgagttttga caaatataaa tataggaatg 1680 tgaatccgaa ggcgcaaata agatgataga gtacctacga gagcttttgt cgaagagcaa 1740 gcctggtgat aagtatggaa gttacgtcct ccagtttgcc gatgattata catacactga 1800 tcctgtagat ggaagtgtag tatcaaaaca aggggttcgg tttgttttca ccgatggttc 1860 aagaattatt taccgtttat caggaacggg ttctgctggt gcaactgtta gagtgtatat 1920 cgaacagttt gaaccagatg tttctaaaca cgacgtcgat gctcaaattg ccttrnacca 1980 tkdaatagat ttagcattat ctgttt 2006 16 451 PRT Pisum sativum 16 Phe Cys Tyr Arg Leu Asp Asn Phe Ile Ile Ser Ala Phe Lys Pro Lys 1 5 10 15 His Ser Asn Val Pro Leu Ser Ile His His Ser Ser Ser Asn Phe Pro 20 25 30 Ser Phe Lys Val Gln Asn Phe Pro Phe Arg Val Arg Tyr Asn Ser Ala 35 40 45 Ile Arg Ala Thr Ser Ser Ser Ser Ser Thr Pro Thr Thr Ile Ala Glu 50 55 60 Pro Asn Asp Ile Lys Ile Asn Ser Ile Pro Thr Lys Pro Ile Glu Gly 65 70 75 80 Gln Lys Thr Gly Thr Ser Gly Leu Arg Lys Lys Val Lys Val Phe Lys 85 90 95 Gln Glu Asn Tyr Leu Ala Asn Trp Ile Gln Ala Leu Phe Asn Ser Leu 100 105 110 Pro Pro Glu Asp Tyr Lys Asn Gly Leu Leu Val Leu Gly Gly Asp Gly 115 120 125 Arg Tyr Phe Asn Lys Glu Ala Ala Gln Ile Ile Ile Lys Ile Ala Ala 130 135 140 Gly Asn Gly Val Gly Lys Ile Leu Val Gly Lys Glu Gly Ile Leu Ser 145 150 155 160 Thr Pro Ala Val Ser Ala Val Ile Arg Lys Arg Glu Ala Asn Gly Gly 165 170 175 Phe Ile Met Ser Ala Ser His Asn Pro Gly Gly Pro Glu Tyr Asp Trp 180 185 190 Gly Ile Lys Phe Asn Tyr Ser Ser Gly Gln Pro Ala Pro Glu Ser Ile 195 200 205 Thr Asp Lys Ile Tyr Gly Asn Thr Leu Ser Ile Ser Glu Ile Lys Ile 210 215 220 Ala Asp Ile Pro Asp Val Asp Leu Ser Asn Val Gly Val Thr Lys Phe 225 230 235 240 Gly Ser Phe Ser Val Glu Val Ile Asp Pro Val Ser Asp Tyr Leu Glu 245 250 255 Leu Leu Glu Thr Val Phe Asp Phe Gln Leu Ile Lys Ser Leu Ile Ser 260 265 270 Arg Pro Asp Phe Arg Phe Thr Phe Asp Ala Met His Ala Val Ala Gly 275 280 285 Ala Tyr Ala Thr Pro Ile Phe Val Asp Lys Leu Gly Ala Ser Pro Asp 290 295 300 Ser Ile Ser Asn Gly Ile Pro Leu Glu Asp Phe Gly His Gly His Pro 305 310 315 320 Asp Pro Asn Leu Thr Tyr Ala Lys Asp Leu Val Asn Ile Met Tyr Ala 325 330 335 Glu Asn Gly Pro Asp Phe Gly Ala Ala Ser Asp Gly Asp Gly Asp Arg 340 345 350 Asn Met Ile Leu Gly Thr Ser Phe Phe Val Thr Pro Ser Asp Ser Val 355 360 365 Ala Val Ile Ala Ala Asn Ala Lys Glu Ala Ile Pro Tyr Phe Lys Asp 370 375 380 Ser Ile Lys Gly Leu Ala Arg Ser Met Pro Thr Ser Gly Ala Leu Asp 385 390 395 400 Arg Val Ala Glu Lys Leu Asn Leu Pro Phe Phe Glu Val Pro Thr Gly 405 410 415 Trp Lys Phe Phe Gly Asn Leu Met Asp Ala Gly Asn Leu Ser Ile Cys 420 425 430 Gly Glu Glu Ser Phe Gly Thr Gly Ser Asp His Ile Arg Glu Lys Asp 435 440 445 Gly Ile Trp 450 17 1055 DNA Pisum sativum CDS (2)..(751) 17 g att gct gat att ccc gat gtt gac tta tca aat gtt gga gtt acg aaa 49 Ile Ala Asp Ile Pro Asp Val Asp Leu Ser Asn Val Gly Val Thr Lys 1 5 10 15 ttc gga agc ttc agt gtg gaa gta ata gac cca gtt tct gat tac ctg 97 Phe Gly Ser Phe Ser Val Glu Val Ile Asp Pro Val Ser Asp Tyr Leu 20 25 30 gag tta ttg gag aca gtg ttc gat ttt cag cta atc aaa agt ctt att 145 Glu Leu Leu Glu Thr Val Phe Asp Phe Gln Leu Ile Lys Ser Leu Ile 35 40 45 tca cgg cca gat ttt agg ttt aca ttt gat gcc atg cat gca gtt gcc 193 Ser Arg Pro Asp Phe Arg Phe Thr Phe Asp Ala Met His Ala Val Ala 50 55 60 ggt gct tat gca aca ccc att ttc gtt gat aaa ctt ggt gct agt ccg 241 Gly Ala Tyr Ala Thr Pro Ile Phe Val Asp Lys Leu Gly Ala Ser Pro 65 70 75 80 gat tca att tca aat gga ata cct ttg gaa gat ttt gga cat ggt cat 289 Asp Ser Ile Ser Asn Gly Ile Pro Leu Glu Asp Phe Gly His Gly His 85 90 95 cct gat cct aat cta aca tac gca aag gat ctt gtc aat att atg tat 337 Pro Asp Pro Asn Leu Thr Tyr Ala Lys Asp Leu Val Asn Ile Met Tyr 100 105 110 gct gaa aac gga cct gat ttt ggt gcc gct agt gat ggt gat ggt gat 385 Ala Glu Asn Gly Pro Asp Phe Gly Ala Ala Ser Asp Gly Asp Gly Asp 115 120 125 aga aat atg att ttg gga aca agt ttc ttc gta act cct tca gac tct 433 Arg Asn Met Ile Leu Gly Thr Ser Phe Phe Val Thr Pro Ser Asp Ser 130 135 140 gta gcc gtt att gca gcc aat gca aaa gaa gcg att ccg tac ttt aag 481 Val Ala Val Ile Ala Ala Asn Ala Lys Glu Ala Ile Pro Tyr Phe Lys 145 150 155 160 gac agt atc aag ggt ctt gca cga tca atg ccg aca agc ggt gct cta 529 Asp Ser Ile Lys Gly Leu Ala Arg Ser Met Pro Thr Ser Gly Ala Leu 165 170 175 gat aga gtt gct gaa aag ttg aac ctc cct ttt ttt gag gtt ccc act 577 Asp Arg Val Ala Glu Lys Leu Asn Leu Pro Phe Phe Glu Val Pro Thr 180 185 190 ggt tgg aaa ttc ttt ggt aat ctt atg gat gct gga aat ctg tcg att 625 Gly Trp Lys Phe Phe Gly Asn Leu Met Asp Ala Gly Asn Leu Ser Ile 195 200 205 tgc ggg gaa gag agt ttt gga aca ggt tct gac cac att cgt gag aaa 673 Cys Gly Glu Glu Ser Phe Gly Thr Gly Ser Asp His Ile Arg Glu Lys 210 215 220 gac gga atc tgc ttg gct ttc gat tat tgc tca ccg caa caa aga cac 721 Asp Gly Ile Cys Leu Ala Phe Asp Tyr Cys Ser Pro Gln Gln Arg His 225 230 235 240 gaa acc agg gga gaa att ggt ctc tgt gtc tgatgttgtg aaggagcatt 771 Glu Thr Arg Gly Glu Ile Gly Leu Cys Val 245 250 gggcaaccta tggtagaaat ttcttttcta gatacgatta cgaggaatgt gaatccgaag 831 gcgcaaataa gatgatagag tacctacgag agcttttgtc gaagagcaag cctggtgata 891 agtatggaag ttacgtcctc cagtttgccg atgattatac atacactgat cctgtagatg 951 gaagtgtagt atcaaaacaa ggggttcggt ttgttttcac cgatggttca agaattattt 1011 accgtttatc aggaacgggt tctgctggtg caactgttag agtg 1055 18 250 PRT Pisum sativum 18 Ile Ala Asp Ile Pro Asp Val Asp Leu Ser Asn Val Gly Val Thr Lys 1 5 10 15 Phe Gly Ser Phe Ser Val Glu Val Ile Asp Pro Val Ser Asp Tyr Leu 20 25 30 Glu Leu Leu Glu Thr Val Phe Asp Phe Gln Leu Ile Lys Ser Leu Ile 35 40 45 Ser Arg Pro Asp Phe Arg Phe Thr Phe Asp Ala Met His Ala Val Ala 50 55 60 Gly Ala Tyr Ala Thr Pro Ile Phe Val Asp Lys Leu Gly Ala Ser Pro 65 70 75 80 Asp Ser Ile Ser Asn Gly Ile Pro Leu Glu Asp Phe Gly His Gly His 85 90 95 Pro Asp Pro Asn Leu Thr Tyr Ala Lys Asp Leu Val Asn Ile Met Tyr 100 105 110 Ala Glu Asn Gly Pro Asp Phe Gly Ala Ala Ser Asp Gly Asp Gly Asp 115 120 125 Arg Asn Met Ile Leu Gly Thr Ser Phe Phe Val Thr Pro Ser Asp Ser 130 135 140 Val Ala Val Ile Ala Ala Asn Ala Lys Glu Ala Ile Pro Tyr Phe Lys 145 150 155 160 Asp Ser Ile Lys Gly Leu Ala Arg Ser Met Pro Thr Ser Gly Ala Leu 165 170 175 Asp Arg Val Ala Glu Lys Leu Asn Leu Pro Phe Phe Glu Val Pro Thr 180 185 190 Gly Trp Lys Phe Phe Gly Asn Leu Met Asp Ala Gly Asn Leu Ser Ile 195 200 205 Cys Gly Glu Glu Ser Phe Gly Thr Gly Ser Asp His Ile Arg Glu Lys 210 215 220 Asp Gly Ile Cys Leu Ala Phe Asp Tyr Cys Ser Pro Gln Gln Arg His 225 230 235 240 Glu Thr Arg Gly Glu Ile Gly Leu Cys Val 245 250 19 1733 DNA Pisum sativum CDS (21)..(1406) 19 cactgttaca gactcgatca atg gct ttc tgt tac aga ctc gac aac ttc atc 53 Met Ala Phe Cys Tyr Arg Leu Asp Asn Phe Ile 1 5 10 atc tct gcg ttt aaa ccc aaa cac tca aat gtc cca ctt tca att cat 101 Ile Ser Ala Phe Lys Pro Lys His Ser Asn Val Pro Leu Ser Ile His 15 20 25 cat tca tca tcc aat ttt cct tct ttc aaa gtt caa aac ttt cct ttc 149 His Ser Ser Ser Asn Phe Pro Ser Phe Lys Val Gln Asn Phe Pro Phe 30 35 40 agg gtt cgc tat aat tca gct att aga gcc act tcg tct tcc tct tct 197 Arg Val Arg Tyr Asn Ser Ala Ile Arg Ala Thr Ser Ser Ser Ser Ser 45 50 55 act ccc aca acc att gca gaa cct aat gac att aag att aac tct att 245 Thr Pro Thr Thr Ile Ala Glu Pro Asn Asp Ile Lys Ile Asn Ser Ile 60 65 70 75 cct act aaa cct att gaa gga caa aaa act ggt acc agt ggc cta aga 293 Pro Thr Lys Pro Ile Glu Gly Gln Lys Thr Gly Thr Ser Gly Leu Arg 80 85 90 aaa aag gtg aaa gtg ttt aag caa gaa aat tac ctt gca aat tgg att 341 Lys Lys Val Lys Val Phe Lys Gln Glu Asn Tyr Leu Ala Asn Trp Ile 95 100 105 cag gca ctg ttt aat tcg ttg ccg ccg gag gat tac aag aat gga ttg 389 Gln Ala Leu Phe Asn Ser Leu Pro Pro Glu Asp Tyr Lys Asn Gly Leu 110 115 120 ttg gtt ttg gga ggc gat ggt cga tac ttc aat aaa gaa gct gca cag 437 Leu Val Leu Gly Gly Asp Gly Arg Tyr Phe Asn Lys Glu Ala Ala Gln 125 130 135 ata ata atc aag att gct gct gga aat ggt gtt gga aaa att ctg gtt 485 Ile Ile Ile Lys Ile Ala Ala Gly Asn Gly Val Gly Lys Ile Leu Val 140 145 150 155 ggg aag gaa ggg ata ttg tca acg cca gcc gtt tct gct gtg ata agg 533 Gly Lys Glu Gly Ile Leu Ser Thr Pro Ala Val Ser Ala Val Ile Arg 160 165 170 aag aga gag gca aat ggt ggg ttt atc atg agt gcg agc cat aac cct 581 Lys Arg Glu Ala Asn Gly Gly Phe Ile Met Ser Ala Ser His Asn Pro 175 180 185 ggt gga cct gaa tat gat tgg ggt att aag ttt aat tac agt agc gga 629 Gly Gly Pro Glu Tyr Asp Trp Gly Ile Lys Phe Asn Tyr Ser Ser Gly 190 195 200 caa cct gca cca gaa tcc atc acc gac aag att tac gga aac acc cta 677 Gln Pro Ala Pro Glu Ser Ile Thr Asp Lys Ile Tyr Gly Asn Thr Leu 205 210 215 tcg att tct gag ata aag att gct gat att ccc gat gtt gac tta tca 725 Ser Ile Ser Glu Ile Lys Ile Ala Asp Ile Pro Asp Val Asp Leu Ser 220 225 230 235 aat gtt gga gtt acg aaa ttc gga agc ttc agt gtg gaa gta ata gac 773 Asn Val Gly Val Thr Lys Phe Gly Ser Phe Ser Val Glu Val Ile Asp 240 245 250 cca gtt tct gat tac ctg gag tta ttg gag aca gtg ttc gat ttt cag 821 Pro Val Ser Asp Tyr Leu Glu Leu Leu Glu Thr Val Phe Asp Phe Gln 255 260 265 cta atc aaa agt ctt att tca cgg cca gat ttt agg ttt aca ttt gat 869 Leu Ile Lys Ser Leu Ile Ser Arg Pro Asp Phe Arg Phe Thr Phe Asp 270 275 280 gcc atg cat gca gtt gcc ggt gct tat gca aca ccc att ttc gtt gat 917 Ala Met His Ala Val Ala Gly Ala Tyr Ala Thr Pro Ile Phe Val Asp 285 290 295 aaa ctt ggt gct agt ccg gat tca att tca aat gga ata cct ttg gaa 965 Lys Leu Gly Ala Ser Pro Asp Ser Ile Ser Asn Gly Ile Pro Leu Glu 300 305 310 315 gat ttt gga cat ggt cat cct gat cct aat cta aca tac gca aag gat 1013 Asp Phe Gly His Gly His Pro Asp Pro Asn Leu Thr Tyr Ala Lys Asp 320 325 330 ctt gtc aat att atg tat gct gaa aac gga cct gat ttt ggt gcc gct 1061 Leu Val Asn Ile Met Tyr Ala Glu Asn Gly Pro Asp Phe Gly Ala Ala 335 340 345 agt gat ggt gat ggt gat aga aat atg att ttg gga aca agt ttc ttc 1109 Ser Asp Gly Asp Gly Asp Arg Asn Met Ile Leu Gly Thr Ser Phe Phe 350 355 360 gta act cct tca gac tct gta gcc gtt att gca gcc aat gca aaa gaa 1157 Val Thr Pro Ser Asp Ser Val Ala Val Ile Ala Ala Asn Ala Lys Glu 365 370 375 gcg att ccg tac ttt aag gac agt atc aag ggt ctt gca cga tca atg 1205 Ala Ile Pro Tyr Phe Lys Asp Ser Ile Lys Gly Leu Ala Arg Ser Met 380 385 390 395 ccg aca agc ggt gct cta gat aga gtt gct gaa aag ttg aac ctc cct 1253 Pro Thr Ser Gly Ala Leu Asp Arg Val Ala Glu Lys Leu Asn Leu Pro 400 405 410 ttt ttt gag gtt ccc act ggt tgg aaa ttc ttt ggt aat ctt atg gat 1301 Phe Phe Glu Val Pro Thr Gly Trp Lys Phe Phe Gly Asn Leu Met Asp 415 420 425 gct gga aat ctg tcg att tgc ggg gaa gag agt ttt gga aca ggt tct 1349 Ala Gly Asn Leu Ser Ile Cys Gly Glu Glu Ser Phe Gly Thr Gly Ser 430 435 440 gac cac att cgt gag aaa gac gga atc tgg aat gtg aat ccg aag gcg 1397 Asp His Ile Arg Glu Lys Asp Gly Ile Trp Asn Val Asn Pro Lys Ala 445 450 455 caa ata aga tgatagagta cctacgagag cttttgtcga agagcaagcc 1446 Gln Ile Arg 460 tggtgataag tatggaagtt acgtcctcca gtttgccgat gattatacat acactgatcc 1506 tgtagatgga agtgtagtat caaaacaagg ggttcggttt gttttcaccg atggttcaag 1566 aattatttac cgtttatcag gaacgggttc tgctggtgca actgttagag tgtatatcga 1626 acagtttgaa ccagatgttt ctaaacacga cgtcgatgct caaattgcct tgaaaccatt 1686 aatagattta gcattatctg tttcaaagct caaagacttc acaggga 1733 20 462 PRT Pisum sativum 20 Met Ala Phe Cys Tyr Arg Leu Asp Asn Phe Ile Ile Ser Ala Phe Lys 1 5 10 15 Pro Lys His Ser Asn Val Pro Leu Ser Ile His His Ser Ser Ser Asn 20 25 30 Phe Pro Ser Phe Lys Val Gln Asn Phe Pro Phe Arg Val Arg Tyr Asn 35 40 45 Ser Ala Ile Arg Ala Thr Ser Ser Ser Ser Ser Thr Pro Thr Thr Ile 50 55 60 Ala Glu Pro Asn Asp Ile Lys Ile Asn Ser Ile Pro Thr Lys Pro Ile 65 70 75 80 Glu Gly Gln Lys Thr Gly Thr Ser Gly Leu Arg Lys Lys Val Lys Val 85 90 95 Phe Lys Gln Glu Asn Tyr Leu Ala Asn Trp Ile Gln Ala Leu Phe Asn 100 105 110 Ser Leu Pro Pro Glu Asp Tyr Lys Asn Gly Leu Leu Val Leu Gly Gly 115 120 125 Asp Gly Arg Tyr Phe Asn Lys Glu Ala Ala Gln Ile Ile Ile Lys Ile 130 135 140 Ala Ala Gly Asn Gly Val Gly Lys Ile Leu Val Gly Lys Glu Gly Ile 145 150 155 160 Leu Ser Thr Pro Ala Val Ser Ala Val Ile Arg Lys Arg Glu Ala Asn 165 170 175 Gly Gly Phe Ile Met Ser Ala Ser His Asn Pro Gly Gly Pro Glu Tyr 180 185 190 Asp Trp Gly Ile Lys Phe Asn Tyr Ser Ser Gly Gln Pro Ala Pro Glu 195 200 205 Ser Ile Thr Asp Lys Ile Tyr Gly Asn Thr Leu Ser Ile Ser Glu Ile 210 215 220 Lys Ile Ala Asp Ile Pro Asp Val Asp Leu Ser Asn Val Gly Val Thr 225 230 235 240 Lys Phe Gly Ser Phe Ser Val Glu Val Ile Asp Pro Val Ser Asp Tyr 245 250 255 Leu Glu Leu Leu Glu Thr Val Phe Asp Phe Gln Leu Ile Lys Ser Leu 260 265 270 Ile Ser Arg Pro Asp Phe Arg Phe Thr Phe Asp Ala Met His Ala Val 275 280 285 Ala Gly Ala Tyr Ala Thr Pro Ile Phe Val Asp Lys Leu Gly Ala Ser 290 295 300 Pro Asp Ser Ile Ser Asn Gly Ile Pro Leu Glu Asp Phe Gly His Gly 305 310 315 320 His Pro Asp Pro Asn Leu Thr Tyr Ala Lys Asp Leu Val Asn Ile Met 325 330 335 Tyr Ala Glu Asn Gly Pro Asp Phe Gly Ala Ala Ser Asp Gly Asp Gly 340 345 350 Asp Arg Asn Met Ile Leu Gly Thr Ser Phe Phe Val Thr Pro Ser Asp 355 360 365 Ser Val Ala Val Ile Ala Ala Asn Ala Lys Glu Ala Ile Pro Tyr Phe 370 375 380 Lys Asp Ser Ile Lys Gly Leu Ala Arg Ser Met Pro Thr Ser Gly Ala 385 390 395 400 Leu Asp Arg Val Ala Glu Lys Leu Asn Leu Pro Phe Phe Glu Val Pro 405 410 415 Thr Gly Trp Lys Phe Phe Gly Asn Leu Met Asp Ala Gly Asn Leu Ser 420 425 430 Ile Cys Gly Glu Glu Ser Phe Gly Thr Gly Ser Asp His Ile Arg Glu 435 440 445 Lys Asp Gly Ile Trp Asn Val Asn Pro Lys Ala Gln Ile Arg 450 455 460 21 35 DNA Pisum sativum 21 ggagatctgg taagcttgtt tttttttttt ttttt 35 

What is claimed is:
 1. A Pisum sativum variety which contains a homozygous bsg gene within its genome, wherein said bsg gene contains a mutation in an intron in a 3′ splice site of said gene.
 2. The Pisum sativum variety of claim 1 wherein the mutation is in a dinucleotide AG, wherein the nucleotide A is replaced by nucleotide T.
 3. A Pisum sativum variety which contains a homozygous bsg gene within its genome, wherein said bsg gene has the nucleotide sequence of SEQ ID NO:1 and said nucleotide sequence contains a mutation in an intron at nucleotide 1594 at the 3′ splice site dinucleotide AG, wherein nucleotide A is replaced with nucleotide T. 